Organoid-derived monolayers and uses thereof

Abstract

The present invention relates to a culture method, in particular a method for obtaining an organoid-derived monolayer, and the use of the organoid-derived monolayer obtained by said method.The present invention also relates to an assay for epithelial barrier function and a method for screening compounds using said assay.

Description

[Technical field]

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

[0002] Technical Field The present invention relates to a culture method, in particular a method for obtaining a two-dimensional organoid-derived monolayer.The present invention also relates to an assay for epithelial barrier function and a method for screening compounds using said assay. [Background technology]

[0003] background There is great interest in epithelial model systems for cellular assays, drug screening, toxicity assays, and the like. Studies of the intestinal epithelium are performed using several in vitro platform systems, such as membrane inserts, organ-on-a-chip systems, Ussing chambers, and intestinal rings. These platforms are suitable for establishing polarized epithelial monolayers with access to both the apical and basal sides of the membrane, using transformed cell lines or primary tissues as models. Transformed intestinal cell lines such as the colorectal (adeno)carcinoma cell lines Caco-2, T84, and HT-29 can differentiate to some extent into polarized intestinal absorptive epithelial cells or mucus-producing cells, but they do not represent the in vivo epithelium due to the absence of some cell types and the aberrant expression of various receptors and transporters (Martinez-Maqueda, D., et al. HT29 Cell Line in The Impact of Food Bio-Actives on Gut Health: In Vitro and Ex Vivo Models. Verhoeckx, K. et al. (eds), Cham (CH): Springer, 113-124 (2015)). In addition, the cell lines are derived from a single donor and therefore do not represent the heterogeneity between patients, and therefore lack complexity and physiological relevance. Primary tissues used in Ussing chambers and as intestinal rings better represent the in vivo situation, but their limited availability, short viability and lack of scalability make them unsuitable tools for high-throughput studies.

[0004] Therefore, there is a need for improved in vitro epithelial model systems. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] The Impact of Food Bio-Actives on Gut Health: In Vitro and Ex Vivo Models. Verhoeckx, K. et al. (eds), Cham (CH): Springer, 113-124 (2015) Summary of the Invention

[0006] The present invention provides a method for obtaining an organoid-derived monolayer, comprising the steps of: i. digesting or dissociating one or more organoids into a suspension of single cells and / or organoid fragments; ii. seeding a semipermeable membrane with the suspension; and iii. Culturing the cells and / or organoid fragments in the presence of growth medium until a monolayer is formed.

[0007] The present invention also provides organoid-derived monolayers obtainable or obtained by the methods provided herein.

[0008] The present invention further provides a method for treating a vascular endothelial cell comprising administering to the patient a therapeutically effective amount of 100 Ω cm 2 The present invention provides an organoid-derived monolayer that is superior in terms of cell viability and cell viability.

[0009] The present invention also provides for the use of the organoid-derived monolayers of the present invention in assays to assess epithelial viability, metabolic activity, permeability, barrier function integrity and / or transport protein activity.

[0010] The present invention further provides a method for identifying a compound capable of modulating epithelial viability, metabolic activity, permeability, barrier function integrity and / or transport protein activity, comprising the steps of: i. contacting an organoid-derived monolayer, such as an organoid-derived monolayer according to the present invention, with one or more candidate molecules; and ii. Assessing the viability, metabolic activity, permeability and / or barrier function integrity of the organoid-derived monolayer and / or the activity of transport proteins in the organoid-derived monolayer.

[0011] The present invention further provides a method for assessing the effect of a compound on epithelial viability, metabolic activity, permeability, barrier function integrity and / or transport protein activity, comprising the steps of: i. contacting an organoid-derived monolayer, such as an organoid-derived monolayer according to the invention, with said compound; and ii. Assessing the viability, metabolic activity, permeability and / or barrier function integrity of the organoid-derived monolayer and / or the activity of transport proteins in the organoid-derived monolayer.

[0012] The present invention further provides a method for identifying mutations associated with epithelial viability, metabolic activity, permeability, barrier function integrity and / or transport protein activity, comprising the steps of: i. assessing the viability, metabolic activity, permeability and / or barrier function integrity of an organoid-derived monolayer, e.g., an organoid monolayer according to the present invention, and / or the activity of transport proteins in the organoid-derived monolayer; ii. Determining the presence of one or more mutations in the genome of one or more cells in the organoid-derived monolayer.

[0013] The present invention further comprises: i. obtaining an organoid-derived monolayer from said human subject using a method of the invention; and ii. Testing the viability, metabolic activity, permeability and / or barrier function integrity of the organoid-derived monolayer and / or the activity of transport proteins in the organoid-derived monolayer. wherein a test result above or below a reference value indicates the presence of said disease or affliction, or an increased risk of said disease or affliction, in a human subject, which affects epithelial viability, metabolic activity, permeability, barrier function integrity and / or transport protein activity, comprising:

[0014] The present invention further provides a method for predicting the likelihood of a patient to respond to a candidate compound, comprising the steps of: i. obtaining an organoid-derived monolayer from said patient using the methods of the invention; ii. contacting the organoid-derived monolayer with the compound; and iii. Assessing the viability, metabolic activity, permeability and / or barrier function integrity of the organoid-derived monolayer and / or the activity of transport proteins in the organoid-derived monolayer.

[0015] Detailed Description Preparation and culture of organoid-derived monolayers Preparation of organoid-derived monolayers The organoid-derived monolayer of the present invention is prepared using organoids. "Organoid" refers to a cellular structure obtained by the proliferation of adult (post-embryonic) epithelial stem cells, preferably characterized by the expression of Lgr5, and composed of tissue-specific cell types that self-organize by cell sorting and spatially restricted lineage commitment (e.g., as described in Clevers, Cell. 2016 Jun 16; 165(7): 1586-1597, see in particular the section entitled "Organoids derived from adult stem cells" starting on page 1590). Methods for obtaining and culturing organoids from various tissues have already been described in WO2010 / 090513, WO2012 / 014076, WO2015 / 173425, WO2016 / 083613 and WO2017 / 220586. Preferably, the adult epithelial stem cells are not derived from induced pluripotent stem (iPS) cells.

[0016] In some embodiments, the organoid-derived monolayers of the present invention comprise: digesting or dissociating one or more organoids into a suspension of single cells and / or organoid fragments; seeding a semipermeable membrane with the suspension; and The cells and / or organoid fragments are obtained by a method comprising the step of culturing the cells and / or organoid fragments in the presence of a growth medium until a monolayer membrane is formed.

[0017] Organoid fragments include any fragment of organoid, such as intestinal crypts from organoid.In some embodiments, organoid fragments are cell clumps, preferably less than 10 cells, less than 5 cells, preferably 2-4 cells.In preferred embodiments, one or more organoids are digested or dissociated into a suspension containing single cells and cell clumps.

[0018] In some embodiments, following digestion or dissociation of one or more organoids, the suspension of single cells and / or organoid fragments is centrifuged and resuspended before seeding on the semipermeable membrane. In some embodiments, the suspension of single cells and / or organoid fragments is centrifuged and resuspended at a cell density suitable for seeding, for example, about 0.5×10 per mL. 6 Cells, approximately 10 per mL 6 Cells, approximately 2 x 10 per mL 6 Cells, approximately 3 x 10 per mL 6 Cells, approximately 4 x 10 per mL 6 Approximately 5 x 10 cells or 1 mL 6 In another embodiment, the suspension of single cells and / or organoid fragments is adjusted to about 0.2×10 cells per mL. 6 Cells, approximately 0.3 x 10 per mL 6 Cells, approximately 0.4 x 10 per mL 6 Cells, approximately 0.5 x 10 per mL 6 Cells, approximately 10 per mL 6 Cells, approximately 2 x 10 per mL 6 Cells, approximately 3 x 10 per mL 6 Cells, approximately 4 x 10 per mL 6 Approximately 5 x 10 cells or 1 mL 6 In some embodiments, the suspension of single cells and / or organoid fragments is adjusted to about 0.2×10 cells per mL before seeding. 6 Less than cells, approximately 0.3 x 10 per mL 6 Less than cells, approximately 0.4 x 10 per mL 6 Less than cells, approximately 0.5 x 10 per mL 6 Less than cells, approximately 10 per mL 6 Less than 2 x 10 cells per mL 6 Less than cells, approximately 3 x 10 per mL 6 Less than 4 x 10 cells per mL 6 Less than or equal to approximately 5 x 10 cells per mL 6 In some embodiments, the suspension of single cells and / or organoid fragments is adjusted to less than about 0.1-1 x 10 cells per mL before seeding. 6Cells, approximately 0.25-0.75 x 10 per mL 6 Cells, approximately 0.3-0.5 x 10 per mL 6 Cells, approximately 0.35-0.45 x 10 per mL 6 Cells, preferably about 0.4 x 10 per mL 6 In some embodiments, the suspension of single cells and / or organoid fragments is adjusted to about 0.5×10 cells per mL before seeding. 6 Less than cells, approximately 0.6 x 10 per mL 6 Less than cells, approximately 0.7 x 10 per mL 6 Less than cells, approximately 0.8 x 10 per mL 6 Less than cells, approximately 0.9 x 10 per mL 6 Less than cells, approximately 10 per mL 6 Less than cells, approximately 1.1 x 10 per mL 6 Less than cells, approximately 1.2 x 10 per mL 6 Less than cells, approximately 1.3 x 10 per mL 6 Less than cells, approximately 1.4 x 10 per mL 6 Less than cells, approximately 1.5 x 10 per mL 6 In some embodiments, the suspension of single cells and / or organoid fragments is adjusted to less than about 0.2×10 cells per mL. 6 Cells, approximately 0.3 x 10 per mL 6 Cells, approximately 0.4 x 10 per mL 6 Cells, approximately 0.5 x 10 per mL 6 Cells, approximately 10 per mL 6 Cells, approximately 1.5 x 10 per mL 6 Cells, approximately 2 x 10 per mL 6 Cells, approximately 3 x 10 per mL 6 Cells, approximately 4 x 10 per mL 6 Approximately 5 x 10 cells or 1 mL 6 In some embodiments, the suspension of single cells and / or organoid fragments is adjusted to about 0.1-5×10 cells per mL. 6 Cells, approximately 0.25-2.5 x 10 per mL 6 Cells, approximately 0.5-1.5 x 10 per mL 6Approximately 0.75-1.25 x 10 cells per mL 6 Cells, approximately 0.8-1.2 x 10 per mL 6 is regulated by cells.

[0019] As shown in the Examples, the seeding density of intestinal cells is preferably about 0.45×10 per mL. 6 The seeding density of lung cells is preferably about 0.4×10 per mL. 6 The seeding density of kidney cells is preferably about 10 per mL. 6 It may be a cell.

[0020] In some embodiments, about 0.1×10 6 cells, approximately 0.2 x 10 6 cells, approximately 0.3 x 10 6 cells, approximately 0.4 x 10 6 cells, approximately 0.5 x 10 6 cells, approximately 0.6 x 10 6 cells, approximately 0.7 x 10 6 cells, approximately 0.7 x 10 6 cells, approximately 0.8 x 10 6 cells, approximately 0.9 x 10 6 cells or about 10 6 Cells are seeded onto the semi-permeable membrane. In some embodiments, about 0.45×10 6 The cells are seeded, for example, on a semi-permeable membrane in a standard 96-well plate.As shown in the examples, this seeding density is particularly suitable for intestinal-derived organoids.

[0021] In some embodiments, less than about 20,000 cells, less than about 30,000 cells, less than about 40,000 cells, less than about 50,000 cells, less than about 60,000 cells, less than about 70,000 cells, less than about 80,000 cells, less than about 90,000 cells, less than about 100,000 cells, or less than about 250,000 cells are seeded onto the semipermeable membrane. In some embodiments, about 30,000 cells, about 40,000 cells, about 50,000 cells, about 60,000 cells, about 70,000 cells, about 80,000 cells, or about 90,000 cells are seeded onto the semipermeable membrane. In some embodiments, about 5,000 to 500,000 cells, about 10,000 to 250,000 cells, about 20,000 to 100,000 cells, about 30,000 to 50,000 cells, about 35,000 to 45,000 cells are seeded on the semipermeable membrane. In some embodiments, about 40,000 cells are seeded on the semipermeable membrane, for example, in a standard 96-well plate. The inventors have unexpectedly found that seeding a smaller number of cells results in higher TEER values ​​for lung monolayers. Thus, in some embodiments, particularly when the organoids are derived from the lung, about 40,000 cells are seeded on the semipermeable membrane, for example, in a standard 96-well plate.

[0022] In some embodiments, less than about 100,000 cells, less than about 150,000 cells, less than about 200,000 cells, or less than about 250,000 cells are seeded onto the semipermeable membrane. In some embodiments, about 30,000 cells, about 40,000 cells, about 50,000 cells, about 60,000 cells, about 70,000 cells, about 80,000 cells, about 90,000 cells, or about 100,000 cells are seeded onto the semipermeable membrane. In some embodiments, about 20,000-500,000 cells, about 30,000-400,000 cells, about 40,000-300,000 cells, about 50,000-250,000 cells, about 60,000-200,000 cells, about 70,000-150,000 cells, about 80,000-120,000 cells are seeded onto the semipermeable membrane. In some embodiments, about 100,000 cells are seeded onto the semipermeable membrane, for example, in a standard 96-well plate. The inventors have unexpectedly found that while seeding higher cell numbers results in higher TEER values ​​for kidney monolayers, this effect plateaus at about 100,000 cells per well of a 96-well plate. Thus, in some embodiments, particularly where the organoids are derived from the kidney, approximately 100,000 cells are seeded onto a semi-permeable membrane, for example, in a standard 96-well plate.

[0023] The growth medium may be any growth medium suitable for epithelial stem or progenitor cells, preferably a growth medium suitable for epithelial stem cells (e.g., as described in WO2010 / 090513, WO2012 / 014076, WO2012 / 168930 or WO2015 / 173425).

[0024] In some embodiments, the growth medium comprises a receptor tyrosine kinase ligand, a BMP inhibitor and a Wnt agonist.

[0025] For example, in some embodiments, the growth medium comprises EGF, Noggin and Wnt conditioned medium, hi some embodiments, the growth medium comprises EGF, Noggin, Respondin and Wnt substitute.

[0026] In some embodiments, the growth medium further comprises nicotinamide and a p38 inhibitor, such as SB202190. In some embodiments, the growth medium further comprises a TGF-beta inhibitor.

[0027] In a preferred embodiment, the growth medium comprises (i) EGF (e.g., at a concentration of about 50 ng / ml), (ii) Noggin (e.g., at a concentration of about 100 ng / ml), (iii) Rspondin (e.g., at a concentration of about 250 ng / mL), (iv) Wnt surrogate (e.g., NGS-Wnt at a concentration of about 0.5 nM), (v) p38 inhibitor (e.g., SB-203580 at a concentration of about 10 μM), (vi) TGF-beta inhibitor (e.g., A83-01 at a concentration of about 500 nM), and (vii) nicotinamide (e.g., at a concentration of about 10 mM).

[0028] In another preferred embodiment, the growth medium comprises (i) EGF (e.g., at a concentration of about 50 ng / ml), (ii) Noggin (e.g., at a concentration of about 100 ng / ml), (iii) Wnt conditioned medium (e.g., about 50% of the final volume), (iv) a p38 inhibitor (e.g., SB-203580 at a concentration of about 10 μM), (v) a TGF-beta inhibitor (e.g., A83-01 at a concentration of about 500 nM), and (vi) nicotinamide (e.g., at a concentration of about 10 mM).

[0029] In some embodiments, particularly when organoid is derived from lung, growth medium comprises one or more receptor tyrosine ligand, Wnt agonist, TGF-beta inhibitor and BMP inhibitor.In some embodiments, growth medium comprises FGF, Rspondin, TGF-beta inhibitor, BMP inhibitor, Rho-kinase inhibitor and p38 inhibitor. In a preferred embodiment, the growth medium comprises (i) FGF (e.g., FGF-7 at a concentration of about 25 ng / ml and FGF-10 at a concentration of about 100 ng / mL), (ii) Rspondin (e.g., Rspondin-3 at a concentration of about 250 ng / mL), (iii) a TGF-beta inhibitor (e.g., A83-01 at a concentration of about 500 nM), (iv) a BMP inhibitor (e.g., Noggin-Fc fusion protein conditioned medium at about 2% of the final volume), (v) a Rho-kinase inhibitor (e.g., Y-27632 at a concentration of about 10 μM), and (vi) a p38 kinase inhibitor (e.g., SB202190 at a concentration of about 500 nM).

[0030] In some embodiments, particularly when organoid is derived from kidney, growth medium comprises one or more receptor tyrosine ligands, Wnt agonist and TGF-beta inhibitor.In some embodiments, growth medium comprises EGF, FGF, Rspondin, TGF-beta inhibitor and Rho-kinase inhibitor.In a preferred embodiment, growth medium comprises (i) EGF (e.g., about 50ng / ml concentration), (ii) FGF (e.g., about 100ng / ml concentration of FGF-10), (iii) Rspondin (e.g., about 10% of final volume of Rspo1-conditioned medium), (iv) TGF-beta inhibitor (e.g., about 500nM concentration of A83-01), and (v) Rho-kinase inhibitor (e.g., about 10μM concentration of Y-27632).

[0031] The present inventors have found that during the stage of culturing cells and / or organoid fragments in the presence of growth medium, the TEER of monolayer increases over time and reaches a plateau when the monolayer reaches confluence.In some embodiments, the monolayer is cultured in the presence of growth medium until the TEER of the monolayer is stable, for example, until the TEER does not increase or decrease by more than 50%, more than 40%, more than 30%, more than 20% or more than 10% in an interval of 24 hours, 2 days, 3 days, 4 days, 5 days or more.For example, the TEER does not increase or decrease by more than 20% in an interval of 24 hours. In some embodiments, the monolayer has a TEER of about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, or about 1000 Ω·cm 2 In some embodiments, the monolayer is cultured in the presence of growth medium until its TEER reaches about 100 Ω cm. 2 The cells are cultured in the presence of growth medium until the cells reach a concentration of 100 mM.

[0032] The inventors have demonstrated that organoid-derived intestinal monolayers further cultured in the presence of differentiation medium are improved since they reach a higher TEER than monolayers cultured only in the presence of growth medium. The higher TEER, in other words, a larger dynamic range, makes such organoid-derived monolayers particularly useful in assays for evaluating, for example, epithelial viability, metabolic activity, permeability, barrier integrity, and / or transport protein activity. Thus, in a preferred embodiment, the method further comprises culturing the monolayer in the presence of differentiation medium. In some embodiments, the TEER of the monolayer is further increased during the step of culturing the monolayer in the presence of differentiation medium. In some embodiments, the TEER of the monolayer is increased to 500 Ω·cm during the step of culturing the monolayer in the presence of differentiation medium. 2 Super, 600Ω cm 2 Super, 700Ω cm 2 Super, 800Ω cm 2 Super, 900Ω cm2 Super, 1000Ω cm 2 Super, 1100Ω cm 2 Super, 1200Ω cm 2 Super, 1300Ω cm 2 Ultra, 1400Ω cm 2 or greater than 1500 Ω cm 2 Reach super.

[0033] The differentiation medium can be any suitable differentiation medium for organoid, for example, as described in WO2015 / 173425, WO2017 / 149025 and WO2017 / 220586.The exemplary differentiation medium that can be used in the present invention is described herein.The desired cell composition in organoid-derived monolayer can be achieved by selecting appropriate differentiation medium.

[0034] The inventors have demonstrated that when organoid-derived monolayers are cultured in a differentiation medium containing a Notch inhibitor, an EGFR pathway inhibitor and a Wnt agonist, the monolayers exhibit higher TEER values ​​and more heterogeneous cellular composition than those achieved in other differentiation media.

[0035] Thus, in preferred embodiments, the method further comprises culturing the monolayer in the presence of a differentiation medium comprising a Notch inhibitor, an EGFR pathway inhibitor and a Wnt agonist. In some ... 2 In some embodiments, the method includes culturing the monolayer in the presence of a differentiation medium comprising a Notch inhibitor, an EGFR pathway inhibitor, and a Wnt agonist until the monolayer reaches a TEER of at least 1000 Ω cm. 2 In some embodiments, the method includes culturing the monolayer in the presence of a differentiation medium comprising a Notch inhibitor, an EGFR pathway inhibitor, and a Wnt agonist until the monolayer reaches a TEER of at least 1500 Ω cm. 2The method includes culturing the monolayer in the presence of a differentiation medium comprising a Notch inhibitor, an EGFR pathway inhibitor, and a Wnt agonist until the monolayer reaches a TEER of 100%. Exemplary differentiation media comprising a Notch inhibitor, an EGFR pathway inhibitor, and a Wnt agonist are those described herein.

[0036] This application exemplifies the preparation and use of organoid-derived monolayer membranes from intestine, lung and kidney.Intestine is epithelial tissue and is part of digestive system.Lung and kidney are also epithelial tissues.Therefore, those skilled in the art should recognize that the method and use described herein can be applied to other epithelial tissues, especially other epithelial tissues of digestive system.

[0037] In some embodiments, organoid-derived monolayer is derived from digestive system.In some embodiments, organoid-derived monolayer is derived from digestive tract.Preferably, organoid-derived monolayer is derived from intestinal tract.

[0038] In some other preferred embodiments, the organoid-derived monolayer is derived from the lung.

[0039] In yet another preferred embodiment, the organoid-derived monolayer is derived from the kidney.

[0040] In some embodiments, the organoid-derived monolayer is derived from a mammal (e.g., human, non-human primate, rat, dog or miniature pig).In some embodiments, the monolayer is derived from a dog.In some embodiments, the monolayer is derived from a rat.Preferably, the monolayer is derived from a human.

[0041] In some embodiments, organoid-derived monolayer is derived from healthy human subjects.In some embodiments, particularly when organoid-derived monolayer is derived from digestive system, organoid-derived monolayer is derived from human with digestive system disease or disorder, such as inflammatory bowel disease (e.g., Crohn's disease (CD) or ulcerative colitis (UC)), celiac disease or leaky gut syndrome.

[0042] Wnt agonists The proliferation medium of the present invention comprises a Wnt agonist. In some embodiments, the differentiation medium of the present invention comprises a Wnt agonist.

[0043] The Wnt signaling pathway and small molecules that activate Wnt signaling are described in Nusse and Clevers (2017, Cell 169(6):985-999). Activation of the Wnt signaling pathway typically inhibits the degradation of β-catenin and enhances β-catenin-mediated signaling. The pathway is defined by a series of events that occur when the cell surface Wnt receptor complex, which includes the Frizzled receptor and LRP5 / 6, is activated by extracellular signaling molecules, typically members of the Wnt family. This activates Dishevelled family proteins, which inhibit the protein destruction complex that degrades intracellular β-catenin. The destruction complex is formed from structural components including APC and axin, to which casein kinases CK1α, δ, and ε, as well as GSK-3, are recruited. The destruction complex is believed to phosphorylate β-catenin and expose it to the ubiquitin ligase β-TrCP. β-catenin is then ubiquitinated and degraded by the proteasome.

[0044] The main effector function of β-catenin is in the nucleus, where it controls transcription through interactions with various transcription factors, including the TCF / LEF family transcription factors (e.g., Tcf-1, Tcf-3, Tcf-4, and Lef1).

[0045] The Wnt pathway is highly regulated. For example, when Rspondin binds to its receptors (Lgr4, Lgr5, and / or Lgr6), Wnt signaling is enhanced. However, it has been shown that two transmembrane E3 ubiquitin ligases, Rnf43 and Znrf3, remove Rspondin receptors (e.g., Lgr4, Lgr5, and / or Lgr6) from the cell surface (see, e.g., de Lau et al. 2016). Rspondin is a vertebrate-specific Wnt enhancer. In addition, the binding of Dishevelled family proteins to Frizzled receptors can be inhibited by Dapper family proteins (e.g., Dapper1 and Dapper3). Furthermore, the activity of the destruction complex is believed to be partially controlled by the phosphorylation status of APC, Axin, and GSK-3. For example, dephosphorylation of APC or axin by phosphatases (serine / threonine phosphatases such as PP1, PP2C, or PP2A) can inhibit β-catenin degradation. Additionally, phosphorylation of GSK-3 by kinases (e.g., p38 MAPK, PKA, PKB, PKC, p90RSK, or p70S6K) can inhibit GSK-3 activity and inhibit β-catenin degradation.

[0046] The stability of the destruction complex is thought to be controlled in part by two PARPs, tankyrase 1 and 2. Poly(ADP-ribosyl)ation and autopoly(ADP-ribosyl)ation of axin by these tankyrases can promote de-oligomerization of the destruction complex.

[0047] In the nucleus, Dishevelled family proteins can form a complex with the histone deacetylase SIRT1, which supports the transcription of Wnt target genes.

[0048] The protein thought to be key in Wnt secretion is the multispanning membrane protein porcupine (Porc), and its deletion results in the accumulation of Wnt in the endoplasmic reticulum.

[0049] A Wnt agonist is defined as an agent that activates TCF / LEF-mediated transcription in cells. Thus, Wnt agonists are selected from true Wnt agonists that bind to and activate the Wnt receptor complex, including any and all Wnt family proteins, inhibitors of intracellular β-catenin degradation, GSK inhibitors (such as CHIR9901), and TCF / LEF activators. In some embodiments, the Wnt agonist is a secreted glycoprotein, including Wnt-1 / Int-1, Wnt-2 / Irp (InM-related protein), Wnt-2b / 13, Wnt-3 / Int-4, Wnt-3a (R&D systems), Wnt-4, Wnt-5a, Wnt-5b, Wnt-6 (Kirikoshi H et al 2001 Biochem Biophys Res Com 283 798-805), Wnt-7a (R&D systems), Wnt-7b, Wnt-8a / 8d, Wnt-8b, Wnt-9a / 14, Wnt- 9b / 14b / 15, Wnt-10a, Wnt-10b / 12, WnM 1, and Wnt-16. An overview of human Wnt proteins is given in "THE WNT FAMILY OF SECRETED PROTEINS", R&D Systems Catalog, 2004. In some embodiments, the Wnt agonist is an inhibitor of RNF43 or ZNRF3. It has been shown that RNF43 and ZNRF3 are present in the cell membrane and that the level of Wnt receptor complex in the membrane is negatively regulated, possibly by ubiquitination of Frizzled. Therefore, the inventors hypothesize that inhibition of RNF43 or ZNRF3 with antagonistic antibodies, RNAi or small molecule inhibitors will indirectly stimulate the Wnt pathway. RNF43 and ZNRF3 have catalytic ring-shaped domains (with ubiquitination activity) that can be targets for designing small molecule inhibitors. Several anti-RNF43 and anti-ZNRF3 antibodies are commercially available. In some embodiments, such antibodies are suitable Wnt agonists in the context of the present invention.

[0050] The Wnt agonist in the growth medium or differentiation medium is preferably any agonist that can stimulate the Wnt pathway via the Lgr5 cell surface receptor, i.e., in a preferred embodiment, the Wnt agonist in the growth medium is an Lgr5 agonist. Known Lgr5 agonists include Rspondin, its fragments and derivatives, and anti-Lgr5 antibodies (see, for example, WO 2012 / 140274, especially Figures 22-24, and De Lau, W. et al. Nature, 2011 Jul 4; 476(7360): 293-7). The preferred Lgr5 agonist is Rspondin. Any suitable Rspondin can be used, for example, selected from one or more of Rspondin 1, Rspondin 2, Rspondin 3 and Rspondin 4 or their derivatives. For example, any of Rspondin 1 (NU206, Nuvelo, San Carlos, CA), Rspondin 2 ((R&D systems), Rspondin 3, and Rspondin-4) can be used. Rspondin can be used at any suitable concentration, for example, at least 100ng / ml, more preferably at least 200ng / ml, more preferably about 250ng / ml. An example of an agonistic anti-Lgr5 antibody is 1D9 (BDB562733, No:562733, available from BD Biosciences). A fragment of Rspondin can be used as a Wnt agonist. For example, in some embodiments, the Wnt agonist is a fragment of Rspondin that includes or consists of a furin domain.

[0051] In some embodiments, the Wnt agonist in the growth or differentiation medium is a Wnt surrogate. The Wnt surrogate is a water-soluble Wnt agonist engineered by linking antagonistic Fzd and Lrp5 / 6 binding modules into a single polypeptide chain, thus forcing receptor heterodimerization while blocking endogenous Wnt binding. The Wnt surrogate supports the growth of a wide range of cultures. Furthermore, the Wnt surrogate is a non-lipidated Wnt agonist that can be produced in serum-free medium and maintained in a frozen state, avoiding the activity differences of Wnt conditioned medium caused by different laboratories (Janda CY, et al. Surrogate Wnt agonists that phenocopy canonical Wnt and β-catenin signalling. Nature. 2017 May 11; 545 (7653): 234-237). In some embodiments, the Wnt surrogate is a next-generation Wnt surrogate (NGS-Wnt), for example as described in Miao, Y. et al. (Next-generation surrogate Wnts support organoid growth and deconvolute Frizzled pleiotropy in vivo. Cell Stem Cell. 27 (5), 840-851 (2020)). NGS-Wnt can be provided at a concentration of about 0.1 nM to about 0.5 nM. In some embodiments, the proliferation medium comprises NGS-Wnt at a concentration of about 0.5 nM. In some embodiments, the differentiation medium comprises NGS-Wnt at a concentration of about 0.1 nM.

[0052] The Wnt agonist is preferably added to the culture medium in an amount that is at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 50%, more preferably at least 70%, more preferably at least 90%, more preferably at least 100% effective in stimulating Wnt activity in cells when evaluated in the same cell type, compared to the level of Wnt activity in the absence of said molecule. As known to those skilled in the art, Wnt activity can be determined by measuring the transcriptional activity of Wnt, for example, by pTOPFLASH and pFOPFLASH Tcf luciferase reporter constructs (Korinek et al., 1997. Science 275:1784-1787). Wnt activity can also be determined using the LEADING LIGHT® Wnt Reporter Assay Starter Kit (Enzo Life Sciences, Catalog No. ENZ-61001-0001), which uses an engineered 3T3 mouse fibroblast cell line expressing a firefly luciferase reporter gene under the control of a Wnt-responsive promoter (TCF / LEF).

[0053] Soluble Wnt agonists such as Wnt-3a may be provided in the form of a Wnt conditioned medium. For example, about 10% to about 50% Wnt conditioned medium may be used.

[0054] Rspondin may be provided in the form of an Rspo conditioned medium. For example, about 10% to about 30% (e.g., about 10 ng / ml to about 10 μg / ml, preferably about 1 μg / ml) of an Rspo conditioned medium may be used.

[0055] Examples of Respondin mimetics suitable for use in the present invention are provided in WO 2012 / 140274, which is incorporated herein by reference.

[0056] One or more Wnt agonists, for example, two, three, four or more Wnt agonists, can be used in the growth medium or differentiation medium. In one embodiment, the medium comprises an Lgr5 agonist (for example, Rspondin) and further comprises an additional Wnt agonist. In this context, the additional Wnt agonist can be selected from the group consisting of, for example, Wnt-3a, GSK inhibitor (such as CHIR99021), Wnt-5, Wnt-6a, Norrin, and NGS-Wnt. In one embodiment, the growth medium or differentiation medium comprises Rspondin and further comprises a soluble Wnt ligand, such as Wnt3a or NGS-Wnt. The addition of a soluble Wnt ligand has been found to be particularly advantageous for the growth of human epithelial stem cells (described in WO2012 / 168930).

[0057] Wnt inhibitors In some embodiments, the differentiation medium of the present invention comprises a Wnt inhibitor. Any suitable Wnt inhibitor may be used.

[0058] The Wnt signaling pathway can be inhibited at many levels, and Wnt inhibitors are reviewed in detail in Voronkov and Krauss (2013) Current Pharmaceutical Design 19:634 664, and Tran and Zheng (2017) Protein Science 26:650-661. Wnt inhibitors are commercially available, for example, from R&D systems, Santa Cruz Biotechnology, and Selleckchem.

[0059] A Wnt inhibitor is defined as an agent that inhibits TCF / LEF-mediated transcription in a cell or cell population. Thus, Wnt inhibitors suitable for use in the present invention include: (1) Wnt secretion inhibitors (e.g., Porc inhibitors such as LGK974, IWP-1 or IWP-2), (2) competitive and noncompetitive inhibitors of the interaction of Wnt or Respondin with their respective receptors (e.g., OMP-18R5, OMP54F28); (3) Factors that promote the degradation of components of the Wnt receptor complex (such as LRP) (e.g., niclosamide), and factors that promote the degradation of Rspondin receptors (such as Znrf3 and / or Rnf43) or factors that activate Znrf3 and / or Rnf43; (4) Inhibitors of Dishevelled family proteins (inhibitors that reduce binding of Dishevelled family proteins to components of the Frizzled receptor and / or destruction complex (e.g., Dapper family proteins, FJ9, Sulindac, 3289 8625, J01-017a, NSC668036) or inhibitors that downregulate the expression of Dishevelled family proteins (e.g., niclosamide), etc.), (5) factors that promote destruction complex activity, including (a) inhibitors of phosphatases (e.g., PP1, PP2A and / or PP2C) that dephosphorylate components of the destruction complex, such as axin and / or APC (e.g., okadaic acid or tautomycin) and (b) inhibitors of kinases (e.g., p38 MAPK, PKA, PKB, PKC, p90RSK or p70S6K) that phosphorylate GSK-3 (e.g., SB239063, SB203580 or Rp-8-Br-cAMP); (6) inhibitors of de-oligomerization of the destruction complex (inhibitors of tankyrase 1 and / or 2 (e.g., XAV939, IWR1, JW74, JW55, 2-[4-(4-fluorophenyl)piperazin-1-yl]-6-methylpyrimidin-4(3H)-one or PJ34), and (7) Inhibitors of β-catenin target gene expression, including inhibitors of the β-catenin:TCF / Lef transcription complex (inhibitors that disrupt the β-catenin:TCF-4 complex (e.g., iCRT3, CGP049090, PKF118310, PKF115-584, ZTM000990, PNU-74654, BC21, iCRT5, iCRT14 or FH535), etc.) and inhibitors of histone deacetylase SIRT1 (e.g., cambinol).

[0060] In some embodiments, the differentiation medium of the present invention comprises a Wnt inhibitor. Any suitable Wnt inhibitor, such as those described in (1) to (7) above, may be used. For example, in a preferred embodiment, the Wnt inhibitor is a Wnt secretion inhibitor, such as a Porc inhibitor (e.g., selected from IWP-2, IWP-1, and LGK974). In another embodiment, the Wnt inhibitor is an inhibitor of β-catenin target gene expression, such as an inhibitor of the β-catenin:TCF / Lef transcription complex or an inhibitor of histone deacetylase SIRT1 (e.g., cambinol). In some embodiments, the inhibitor of the β-catenin:TCF / Lef transcription complex is an inhibitor that disrupts the β-catenin:TCF-4 complex, e.g., an inhibitor selected from iCRT3, CGP049090, PKF118310, PKF115-584, ZTM000990, PNU-74654, BC21, iCRT5, iCRT14, and FH535.

[0061] In some embodiments, the Wnt inhibitor is selected from IWP-2, OMP-18R5, OMP54F28, LGK974, 3289-8625, FJ9, NSC 668036, IWR1, and XAV939.

[0062] In some embodiments, the Wnt inhibitor is selected from iCRT3, PFK115-584, CGP049090, iCRT5, iCRT14, and FH535.

[0063] In some embodiments, the Wnt inhibitor is one of the compounds listed in Table 1 below.

[0064] (Table 1) Wnt inhibitors TIFF2024525079000001.tif63148TIFF2024525079000002.tif236148TIFF2024525079000003.tif243148TIFF20245250790 00004.tif237148TIFF2024525079000005.tif213148TIFF2024525079000006.tif212148TIFF2024525079000007.tif162148

[0065] Other Wnt inhibitors suitable for use in the present invention include TMEM88, KY-02061, KY-02327, BMD4702, DK-520, pyrvinium, derricin, derricidin, carnosic acid, windorphen, IWP-L6, Wnt-C59, ETC-159, E7449 and WIKI4.

[0066] In some embodiments, the differentiation medium of the present invention comprises one or more of TMEM88, KY-02061, KY-02327, BMD4702, DK-520, pyrvinium, derricin, derricidin, carnosic acid, windorphen, IWP-L6, Wnt-C59, ETC-159, E7449, WIKI4 or any of the Wnt inhibitors listed in Table 1.

[0067] The Wnt inhibitor is preferably added to the culture medium in an amount that is at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 50%, more preferably at least 70%, more preferably at least 90%, more preferably 100% effective in inhibiting Wnt activity in cells when evaluated in the same cell type, compared to the level of Wnt activity in the absence of the molecule. As known to those skilled in the art, Wnt activity can be determined by measuring the transcriptional activity of Wnt, for example, by pTOPFLASH and pFOPFLASH Tcf luciferase reporter constructs (Korinek et al. (1997) Science 275:1784-1787). Wnt activity can also be determined using the LEADING LIGHT® Wnt Reporter Assay Starter Kit (Enzo Life Sciences, Catalog No. ENZ-61001-0001), which uses an engineered 3T3 mouse fibroblast cell line expressing a firefly luciferase reporter gene under the control of a Wnt-responsive promoter (TCF / LEF). In addition, Grimaldi et al. (Frontiers in Pharmacology 9:1160) describes a cell model suitable for high-throughput screening of Wnt inhibitors, including DLD-1 cells stably transfected with a luciferase TCF reporter plasmid. Furthermore, cell-based HTRF (homogeneous time-resolved fluorescence) assays for Wnt signaling activity that detect protein levels and phosphorylation of GSK3 and β-catenin are described in Romier et al. ("New cell-based HTRF® assays for the exploration of Wnt signalling pathway" Cisbio Bioassays). Thus, novel Wnt inhibitors can be readily identified by those of skill in the art using assays known in the art.

[0068] In some embodiments, the differentiation medium of the present invention contains a Wnt inhibitor at a concentration of 0.01 to 150 μM, 0.1 to 150 μM, 0.5 to 100 μM, 0.1 to 100 μM, 0.5 to 50 μM, 1 to 100 μM, or 10 to 80 μM, 1 to 20 μM, or 1 to 5 μM.

[0069] In some embodiments, the differentiation medium of the present invention contains IWP-2 at a concentration of 0.01 to 150 μM, 0.1 to 100 μM, 0.5 to 50 μM, 1 to 20 μM, or 1 to 5 μM. For example, in some embodiments, the differentiation medium of the present invention contains IWP-2 at a concentration of about 1.5 μM.

[0070] In some embodiments, the differentiation medium does not contain a Wnt agonist that binds to and activates the Wnt receptor complex, including any and all Wnt family proteins and Rspondins.

[0071] In other embodiments, the differentiation medium further comprises a Wnt agonist, such as R-spondin 1-4 or a biologically active fragment or variant thereof. As described above, R-spondin enhances Wnt signaling at cell surface receptors. It is hypothesized that some Wnt signaling may be required to direct cells to the secretory lineage (rather than the absorptive lineage). Thus, in some embodiments, the differentiation medium comprises both a Wnt agonist (particularly R-spondin) and a Wnt inhibitor. For example, in some embodiments, the differentiation medium comprises R-spondin and a Porc inhibitor (such as IWP-2). In some embodiments, R-spondin is used at a final concentration of 1-1000 ng / ml, 50-1000 ng / ml, or 100-1000 ng / ml. In a preferred embodiment, R-spondin is used at a final concentration of about 250 ng / ml.

[0072] Receptor tyrosine kinase ligands In some embodiments, the proliferation or differentiation medium of the present invention further comprises a receptor tyrosine kinase ligand.

[0073] Receptor tyrosine kinases (RTKs) are high-affinity cell surface receptors for polypeptide growth factors, cytokines, and hormones. RTKs and their ligands are described in detail in Trenker and Jura (Current Opinion in Cell Biology 2020,63:174-185). RTKs are important regulators of cell maintenance, growth, and development, and also play a critical role in the development and progression of many types of cancer. RTK activity can be measured using the Proteome Profiler Human Phospho-RTK Array Kit (R&D systems), which measures the relative phosphorylation of 49 human RTKs.

[0074] In the context of the present invention, a receptor tyrosine kinase ligand is any ligand that activates RTK. Many receptor tyrosine kinase ligands are mitogenic growth factors. Thus, in some embodiments, one or more receptor tyrosine kinase ligands in differentiation medium include one or more mitogenic growth factors.

[0075] There are approximately 20 different known classes of RTKs, including RTK class I (EGF receptor family) (ErbB family), RTK class II (insulin receptor family), RTK class III (PDGF receptor family), RTK class IV (FGF receptor family), RTK class V (VEGF receptor family), RTK class VI (HGF receptor family), RTK class VII (Trk receptor family), RTK class VIII (Eph receptor family), RTK class IX (AXL receptor family), RTK class X (LTK receptor family), RTK class XI (TIE receptor family), RTK class XII (ROR receptor family), RTK class XIII (DDR receptor family), RTK class XIV (RET receptor family), RTK class XV (KLG receptor family), RTK class XVI (RYK receptor family), and RTK class XVII (MuSK receptor family). In some embodiments, the one or more receptor tyrosine kinase ligands include ligands for one or more, or all, of these 20 classes of RTKs.

[0076] RTK class I includes, for example, EGFR / ErbB1, ErbB2 / HER2 / neu, ErbB3 / HER3 and ErbB4 / HER4. RTK class I family ligands include EGF (ErbB1 ligand) and neuregulin (ErbB3 / 4 ligand), which have been shown to be useful in organoid culture (see, for example, WO / 2016 / 083613). RTK class IV (FGF receptor family) and RTK class VI (HGF receptor family) ligands, as well as RTK class II (insulin receptor family) ligands, have also been shown to be useful in organoid culture. Thus, in some embodiments, the one or more receptor tyrosine kinase ligands include ligands for one or more of RTK class I, RTK class II, RTK class IV or RTK class VI.

[0077] In some embodiments, the receptor tyrosine kinase ligand in the growth medium or differentiation medium is selected from the group consisting of epidermal growth factor (EGF), neuregulin, fibroblast growth factor (FGF), hepatocyte growth factor (HGF), and insulin-like growth factor (IGF). In some embodiments, the receptor tyrosine kinase ligand in the growth medium or differentiation medium is selected from the group consisting of epidermal growth factor (EGF), neuregulin, fibroblast growth factor (FGF), and hepatocyte growth factor (HGF). Preferably, the receptor tyrosine kinase ligand is EGF. The receptor tyrosine kinase ligand may be used at any suitable concentration, for example, EGF may be used at a concentration of about 50 ng / mL.

[0078] BMP inhibitors In some embodiments, the differentiation medium of the present invention further comprises a BMP inhibitor.

[0079] BMPs are signaling small molecules that bind to two classes of cell surface bone morphogenetic protein receptors (BMPR-I and BMPRII). The BMPR-I receptor class consists of three receptor types: activin receptor-like kinase-2 (ALK-2 or ActR-IA), ALK-3 (BMPR-IA) and ALK-6 (BMPR-IB). The BMPR-II receptor class consists of three receptor types: BMPR-II, ActR-IIA and ActR-IIB. Upon binding of BMPs, a heterotetrameric complex is formed, containing two type I receptors and two type II receptors. Each BMP receptor contains an intracellular serine / threonine kinase domain in addition to an extracellular binding domain. Following BMP binding, a constitutively active type II receptor kinase phosphorylates the type I receptor kinase domain, which then phosphorylates BMP-responsive SMADs 1, 5 and 8. These BMP-responsive SMADs 1, 5 and 8 can enter the cell nucleus and function as transcription factors. Phosphorylation of these specific SMADs results in various cellular effects, including growth regulation and differentiation. A BMP inhibitor is any inhibitor that significantly reduces signaling through these pathways. For example, a BMP inhibitor can disrupt the interaction of BMP with BMP receptor, bind to BMP receptor and inhibit activation of downstream signaling, inhibit phosphorylation of Smad 1, Smad 5 or Smad 8, inhibit translocation of Smad 1, Smad 5 or Smad 8 to the nucleus, inhibit SMAD 1, SMAD 5 or SMAD 8 mediated transcription of target genes, or inhibit expression, folding or secretion of BMP. In some embodiments, a BMP inhibitor reduces signaling through the BMPR-I receptor class. In some embodiments, a BMP inhibitor reduces signaling through the BMPR-II receptor class. In some embodiments, a BMP inhibitor reduces signaling through SMAD 1 / 5 / 8. Inhibition can be direct or indirect.

[0080] Many BMP inhibitors are known in the art and are disclosed, for example, in Cuny, et al., (2008) Structure-activity relationship study of bone morphogenetic protein (BMP) signaling inhibitors. Bioorg Med Chem Lett 18: 4388-4392 and Sachez-Duffhues (2020) Bone 138: 115472. Any of these BMP inhibitors are suitable for use in the methods of the present invention. Methods for identifying suitable BMP inhibitors are known in the art. A suitable assay is described in Zilberberg et al., BMC Cell Biology 2007 8: 41. Another suitable assay for BMP inhibitors (especially BMP inhibitors that inhibit phosphorylation of Smad 1, 5 or 8 via ALK2 and ALK3) is the cytobot cellular ELISA assay described in Cuny, et al., (2008) Structure-activity relationship study of bone morphogenetic protein (BMP) signaling inhibitors. Bioorg Med Chem Lett 18: 4388-4392, which can be used to identify BMP inhibitors. Further assays for BMP inhibitors are described in Dinter et al. (2019) Methods Mol Biol 1891: 221-233.

[0081] In some embodiments, the BMP inhibitor is selected from noggin, chordin, follistatin, gremlin, tsg (twisted gastrulation), sog (short gastrulation), dorsomorphin, and LDN 193189. In some embodiments, the BMP inhibitor is selected from: a. dorsomorphin or LDN193189 or an analog or variant thereof, and / or b. noggin, sclerostin, chordin, CTGF, follistatin, gremlin, tsg, sog or analogs or variants thereof.

[0082] In a preferred embodiment, the BMP inhibitor is Noggin. Noggin is particularly suitable for in vitro culture methods. Preferably, Noggin is recombinant Noggin.

[0083] In some embodiments, Noggin is included in the proliferation or differentiation medium at a final concentration of 1-1000ng / ml, 10-1000ng / ml, 100-1000ng / ml, 1-500ng / ml, 1-200ng / ml, 1-100ng / ml, 10-500ng / ml, 20-500ng / ml, 10-200ng / ml, 20-200ng / ml, 50-500ng / ml, or 50-200ng / ml. In a preferred embodiment, Noggin is included in the proliferation or differentiation medium at a final concentration of about 100ng / ml.

[0084] In some embodiments, Noggin is provided in the form of Noggin conditioned medium (e.g., Noggin-Fc Fusion Protein conditioned medium (U-Protein Express, Catalog No. N002)). In some embodiments, the proliferation or differentiation medium comprises Noggin conditioned medium at a final concentration of 0.1-10% or 0.5-5%. In preferred embodiments, the proliferation or differentiation medium comprises Noggin conditioned medium at a final concentration of about 1-2%.

[0085] BMP pathway activators In some embodiments, the differentiation medium includes a BMP pathway activator. In some embodiments, the differentiation medium does not include a BMP inhibitor (e.g., noggin). In some embodiments, the differentiation medium includes a BMP pathway activator and does not include a BMP inhibitor (e.g., noggin).

[0086] Methods for identifying suitable BMP pathway activators are known in the art. A suitable assay for measuring BMP activity is described in Zilberberg et al., BMC Cell Biology 2007 8:41.

[0087] In some embodiments, the BMP pathway activator is selected from BMP7, BMP4 and BMP2. Preferred is BMP4.

[0088] In some embodiments, the BMP pathway activator such as BMP4 is present in the differentiation medium at at least 0.01 ng / ml, at least 0.1 ng / ml, at least 1 ng / ml, at least 10 ng / ml, at least 20 ng / ml, at least 25 ng / ml, at least 100 ng / ml, at least 500 ng / ml, at least 1 μg / ml, at least 10 μg / ml, or at least 50 μg / ml. In some embodiments, the BMP pathway activator such as BMP4 is present in the differentiation medium at about 0.01 ng / ml to about 500 ng / ml, about 1 ng / ml to about 500 ng / ml, about 10 ng / ml to about 500 ng / ml, or about 20 ng / ml to about 500 ng / ml. In some embodiments, the BMP pathway activator such as BMP4 is present in the differentiation medium at about 0.01 ng / ml to about 200 ng / ml, about 0.1 ng / ml to about 100 ng / ml, or about 1 ng / ml to about 100 ng / ml. In some embodiments, a BMP pathway activator, such as BMP4, is present in the differentiation medium at about 10 ng / ml.

[0089] In some embodiments, the differentiation medium does not include a BMP pathway activator.

[0090] Notch inhibitors In some embodiments, the differentiation medium comprises a Notch inhibitor. Any suitable Notch inhibitor may be used.

[0091] Notch is a transmembrane surface receptor that can be activated through multiple proteolytic cleavages. One of these is by a protein complex with protease activity called gamma secretase. Gamma secretase is a protease that exerts its cleavage activity within the membrane. Gamma secretase is a complex enzyme that is composed of at least four different proteins, namely presenilin (presenilin 1 or 2), nicastrin, PEN-2, and APH-I. Presenilin is the catalytic center of gamma secretase. Upon ligand binding, the conformation of the Notch receptor changes and the ectodomain is shed through the action of the metalloprotease ADAM protease. This is immediately followed by the action of the gamma secretase complex to release the Notch intracellular domain (NICD). NICD translocates to the nucleus, where it interacts with CSL (C promoter-binding factor / recombinant signal sequence-binding protein Jκ / Supressor-of-Hairless / Lagl). NICD binding converts CSL from a transcriptional repressor to a transcriptional activator, resulting in the expression of Notch target genes.

[0092] In some embodiments, the Notch inhibitor is an inhibitor that can attenuate ligand-mediated activation of Notch (e.g., via a dominant-negative ligand of Notch, or via a dominant-negative Notch, or via an antibody that can at least partially block the interaction of the Notch ligand with Notch), or an inhibitor of an ADAM protease.

[0093] In some embodiments, the Notch inhibitor is a gamma secretase inhibitor, such as DAPT, dibenzazepine (DBZ), benzodiazepine (BZ) or LY 411575. One or more Notch inhibitors may be used, such as two, three, four or more.

[0094] In some embodiments, the Notch inhibitor (e.g., DAPT) is used at a concentration of 0.001 to 200 mM, 0.01 to 100 mM, 0.1 to 50 mM, 0.1 to 20 mM, 0.5 to 10 mM, or 0.5 to 5 mM. In some embodiments, the differentiation medium contains DAPT at a concentration of about 10 μM.

[0095] Notch inhibitors are commercially available, for example, from MedChemExpress. Additional Notch inhibitors can be identified by assaying Notch signaling activity, for example, using the Notch1 Pathway Reporter Kit (BPS Bioscience) or TaqMan™ Array Human Notch Signaling plate (Applied Biosystems).

[0096] EGFR pathway inhibitors In some embodiments, the differentiation medium of the present invention comprises an EGFR pathway inhibitor. Any suitable inhibitor as defined herein may be used.

[0097] The epidermal growth factor receptor (EGFR), also known as ErbB1 or HER1, is a cell surface receptor for members of the epidermal growth factor (EGF) family of extracellular protein ligands. EGFR belongs to the HER family of receptors, which includes four related proteins: EGFR (HER1 / ErbB1), ErbB2 (HER2), ErbB3 (HER3), and ErbB4 (HER4). HER receptors are known to be activated by binding to a variety of ligands, including EGF, TGFA, heparin-binding EGF-like growth factor, amphiregulin, betacellulin, and epiregulin. After ligand binding to the receptor's extracellular domain, the receptor forms a functionally active dimer (EGFR-EGFR (homodimer) or EGFR-HER2, EGFR-HER3, EGFR-HER4 (heterodimer)). Dimerization induces activation of the tyrosine kinase domain, which results in autophosphorylation of the receptor on multiple tyrosine residues. This recruits various adaptor proteins (SHC, GRB2, etc.) and activates a series of intracellular signaling cascades that affect gene transcription.

[0098] The pathways mediating the downstream effects of EGFR have been well studied, and three major signaling pathways have been identified. The first pathway involves the RAS-RAF-MAPK pathway, in which phosphorylated EGFR recruits guanine-nucleotide exchange factors via GRB2 and Shc adaptor proteins, which activates RAS and subsequently stimulates the RAF and MAP kinase pathways, which act on cell proliferation, tumor invasion, and metastasis. Activated RAS activates the protein kinase activity of RAF kinase. RAF kinase phosphorylates and activates MEK (also known as MAP2K or MAPKK), which in turn phosphorylates and activates MAP kinase (ERK, also known as extracellular signal-regulated kinase). The second pathway involves the PI3K / AKT pathway, which activates key cell survival and anti-apoptotic signals through the activation of nuclear transcription factors such as NFKB. The third pathway involves the JAK / STAT pathway, which is also involved in the transcriptional activation of genes related to cell survival. EGFR activation also leads to the phosphorylation of PLCG and the subsequent hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG), thereby activating protein kinase C (PRKC) and CAMK.

[0099] EGFR inhibitors such as anti-EGFR monoclonal antibodies and small molecule EGFR tyrosine kinase inhibitors are available. Some anti-EGFR antibodies, such as cetuximab and panitumumab, bind to the extracellular domain of EGFR monomers and compete with receptor binding by endogenous ligands. In this way, the antibodies block ligand-induced receptor activation. Some small molecule EGFR inhibitors, such as erlotinib, gefitinib, and lapatinib, compete with ATP to bind to the kinase domain of EGFR, thus inhibiting EGFR autophosphorylation and downstream signaling.

[0100] The EGFR signaling pathway and many EGFR inhibitors are described in Singh et al. (2016) Mini-Reviews in Medicinal Chemistry 16:1134-1166. Additional EGFR inhibitors can be identified by assaying EGFR signaling activity, for example, using the EGFR Kinase Assay Kit (BPS Bioscience).

[0101] One or more, for example, two, three, four or more, EGFR pathway inhibitors may be used.

[0102] The EGFR pathway inhibitor is preferably added to the culture medium in an amount that is at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 50%, more preferably at least 70%, more preferably at least 90%, more preferably 100% effective to inhibit EGFR pathway activity in cells when evaluated in the same cell type, compared to the level of EGFR pathway activity in the absence of the molecule. As known to those skilled in the art, EGFR pathway activity can be measured in a variety of ways. For example, an assay for monitoring EGFR activity and inhibitor sensitivity is described in Ghosh et al. (2013) Assay and Drug Development Technologies 11(1):44-51. This particular assay requires a peptide substrate covalently immobilized on magnetic beads. After the kinase reaction, the beads are washed and phosphorylation of the peptide is detected by chemiluminescence using an HRP-conjugated primary antibody against phosphorylated tyrosine. The measured fluorescence intensity is directly proportional to the phosphorylation of the substrate, which is in turn proportional to the EGFR kinase activity. This assay can also be used to screen for inhibitors of other kinases in the EGFR pathway (e.g., RAS, RAF, MEK, or ERK). An alternative method for assaying kinase activity is P 32The assay involves detecting the incorporation of a terminal phosphate from ATP labeled with EGFR. Thus, novel EGFR pathway inhibitors can be readily identified by those of skill in the art using assays known in the art.

[0103] In some embodiments, the EGFR pathway inhibitor is an EGFR inhibitor that inhibits EGFR kinase activity by at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 50%, more preferably at least 70%, more preferably at least 90%, and more preferably 100%.

[0104] In some embodiments, the EGFR pathway inhibitor is a RAS inhibitor that inhibits RAS kinase activity by at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 50%, more preferably at least 70%, more preferably at least 90%, more preferably 100%.

[0105] In some embodiments, the EGFR pathway inhibitor is a RAF inhibitor that inhibits RAF kinase activity by at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 50%, more preferably at least 70%, more preferably at least 90%, and more preferably 100%.

[0106] In some embodiments, the EGFR pathway inhibitor is a MEK inhibitor that inhibits MEK kinase activity by at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 50%, more preferably at least 70%, more preferably at least 90%, and more preferably 100%.

[0107] In some embodiments, the EGFR pathway inhibitor is an ERK inhibitor that inhibits ERK kinase activity by at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 50%, more preferably at least 70%, more preferably at least 90%, and more preferably 100%.

[0108] In some embodiments, EGF is present in the differentiation medium at a concentration of less than 1 mM.

[0109] In some embodiments, the EGFR pathway inhibitor is gefitinib (Santa Cruz Biotechnology), AG-18, AG-490 (tyrophostin B42), AG-1478 (tyrophostin AG-1478), AZ5104, AZD3759, brigatinib, erlotinib, cetuximab, CL-387785 (EKI-785), CNX-2006, icotinib, necitumumab, osimertinib (AZD9291), OSI-420, PD153035 EGFR inhibitors such as HCl, PD168393, pelitinib (EKB-569), rociletinib (CO-1686, AVL-301), TAK-285, tyrophostin 9, vandetanib, WHI-P154, WZ3146, WZ4002, WZ8040, panitumumab, zalutumumab, nimotuzumab, or matuzumab. In some embodiments, the EGFR inhibitor binds to the extracellular domain of the EGFR monomer and competes with receptor binding by EGF. In some embodiments, the EGFR inhibitor binds to the kinase domain of EGFR in competition with ATP. One or more EGFR inhibitors may be used, for example, two, three, four, or more.

[0110] In some embodiments, the EGFR pathway inhibitor is an EGFR and ErbB-2 inhibitor, such as afatinib (Selleckchem), afatinib maleate, AC480 (BMS-599626), AEE788 (NVP-AEE788), AST-1306, canertinib, CUDC-101, dacomitinib, lapatinib, neratinib, poziotinib (HM781-36B), sapitinib (AZD8931) or varlitinib. One or more EGFR and ErbB-2 inhibitors may be used, e.g., two, three, four or more EGFR and ErbB-2 inhibitors.

[0111] In some embodiments, the EGFR pathway inhibitor is an inhibitor of the RAS-RAF-MAPK pathway. In some embodiments, the EGFR pathway inhibitor is an inhibitor of the PI3K / AKT pathway. In some embodiments, the EGFR pathway inhibitor is an inhibitor of the JAK / STAT pathway.

[0112] In some embodiments, the EGFR pathway inhibitor is a RAF inhibitor such as GW5074, ZM 336372, NVP-BHG712, TAK-632, darafenib (GSK2118436), sorafenib, sorafenib tosylate, PLX-4720, AZ 628, CEP-32496 or vemurafenib (PLX4032, RG7204).

[0113] In some embodiments, the EGFR pathway inhibitor is a MEK inhibitor, such as PD0325901 (Sigma Aldrich). In some embodiments, the EGFR pathway inhibitor is an ERK inhibitor, such as SCH772984 (Selleckchem).

[0114] In some embodiments, the EGFR pathway inhibitor is used at a concentration of 0.01 to 200 μM, 0.01 to 100 μM, 0.1 to 50 μM, or 0.1 to 20 μM. For example, in some embodiments, the differentiation medium contains PD0325901 at a concentration of about 100 nM.

[0115] Base medium The proliferation and differentiation media of the present invention include basal media, which is any basal medium suitable for animal or human cells, subject to the limitations set forth herein.

[0116] Basal media for animal or human cell cultures usually contain a number of components necessary to support the maintenance of cultured cells. Appropriate combinations of components can be easily prepared by those skilled in the art, taking into account the following disclosure. Basal media for use in the present invention will generally comprise a nutrient solution containing standard cell culture components such as amino acids, vitamins, lipid supplements, inorganic salts, carbon energy sources, and buffers, as described in more detail in the literature and above. In some embodiments, the culture solution is further supplemented with one or more standard cell culture components selected from, for example, amino acids, vitamins, lipid supplements, inorganic salts, carbon energy sources, and buffers.

[0117] Those skilled in the art will understand from common general knowledge what type of culture medium may be used as the base medium of the proliferation or differentiation medium of the present invention. Potentially suitable cell culture media are commercially available and include, but are not limited to, Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), Knockout-DMEM (KO-DMEM), Glasgow Minimum Essential Medium (G-MEM), Basal Eagle Medium (BME), DMEM / Ham's F12, Advanced DMEM / Ham's F12, Iscove's Modified Dulbecco's Medium and Minimum Essential Medium (MEM), Ham's F-10, Ham's F-12, 199 medium, and RPMI 1640 medium.

[0118] For example, the basal medium may be selected from DMEM / F12 and RPMI 1640 supplemented with glutamine, insulin, penicillin / streptomycin and transferrin. In a further preferred embodiment, Advanced DMEM / F12 or Advanced RPMI is used, which is optimized for serum-free culture and already contains insulin. In this case, the Advanced DMEM / F12 or Advanced RPMI medium is preferably supplemented with glutamine and penicillin / streptomycin. Also preferred is AdDMEM / F12 (Invitrogen) supplemented with N2 and B27. Preferably, the basal medium is Advanced DMEM / F12. More preferably, the basal medium contains Advanced DMEM / F12, glutamine and B27.

[0119] In a preferred embodiment, the basal medium comprises Advanced DMEM / F12, HEPES, penicillin / streptomycin, glutamine, N-acetylcysteine ​​and B27.

[0120] In a more preferred embodiment, the basal medium comprises or consists of Advanced DMEM / F12 supplemented with penicillin / streptomycin, 10 mM HEPES, Glutamax, B27 (all from Life Technologies, Carlsbad, Calif.) and about 1.25 mM N-acetylcysteine ​​(Sigma).

[0121] More preferably, the basal medium is supplemented with purified, natural, semi-synthetic and / or synthetic growth factors and does not contain undefined components such as fetal bovine serum or fetal bovine serum. A variety of different serum replacement preparations are commercially available and known to those skilled in the art. When serum replacement is used, it may be used at about 1% to about 30% of the medium volume according to conventional techniques.

[0122] The growth and differentiation media used in the present invention may contain serum or may be serum and / or serum substitute-free as described elsewhere herein. Preparation of culture media and cells is preferably a GMP process to ensure product consistency, in line with standards required by the FDA for biological products.

[0123] In a preferred embodiment, the proliferation and differentiation medium is feeder cell-free and / or feeder cell conditioned medium-free.

[0124] In a preferred embodiment, the proliferation and differentiation media are free of undefined components.

[0125] In a preferred embodiment, when culturing human cells, human growth factors are used to avoid any foreign contamination. For example, human cultures that do not contain any non-human animal components are also known as xeno-free. In a preferred embodiment, the proliferation medium and / or differentiation medium are xeno-free.

[0126] The growth and differentiation media of the present invention are usually prepared in deionized distilled water. The growth and differentiation media of the present invention will typically be sterilized before use, for example by ultraviolet light, heating, irradiation or filtration to prevent contamination. The growth and differentiation media can be frozen (e.g., at -20°C or -80°C) for storage or transportation. The media can contain one or more antibiotics to prevent contamination. The media can have an endotoxin content of less than 0.1 endotoxin units per ml, or less than 0.05 endotoxin units per ml. Methods for measuring the endotoxin content of cultures are known in the art.

[0127] The preferred base medium is a defined synthetic medium buffered with a carbonate-based buffer to pH 7.4 (preferably pH 7.2-7.6, or at least 7.2 and no more than 7.6), and the cells are cultured in an atmosphere containing 5%-10% CO2, or at least 5% and no more than 10% CO2, preferably 5% CO2.

[0128] additional factor p38 MAPK inhibitors In some embodiments of the present invention, the growth medium further comprises a p38 MAPK inhibitor (also referred to herein as a p38 inhibitor, meaning any inhibitor that directly or indirectly negatively regulates p38 signaling). In some embodiments, the inhibitor according to the present invention binds to p38 (GI number 1432) and reduces its activity. p38 protein kinase is a member of the mitogen-activated protein kinase (MAPK) family. MAPKs are serine / threonine-specific protein kinases that respond to extracellular stimuli such as environmental stress and inflammatory cytokines and regulate various cellular activities such as gene expression, mitosis, differentiation, proliferation, and cell survival / apoptosis. p38 MAPK exists as α, β, β2, γ, and δ isoforms. A p38 inhibitor is an agent that binds to at least one p38 isoform and reduces its activity. Various methods are known for determining whether a substance is a p38 inhibitor and may be used in combination with the present invention. Examples include phospho-specific antibody detection of phosphorylation at Thr180 / Tyr182 (which provides a highly accurate measure of cellular p38 activation or inhibition), biochemical recombinant kinase assays, tumor necrosis factor alpha (TNFα) secretion assays, and the DiscoverRx high-throughput screening platform for p38 inhibitors (see http: / / www.discoverx.com / kinases / literature / biochemical / collaterals / DRx_poster_p38%20KBA.pdf). Several p38 activity assay kits also exist (e.g., Millipore, Sigma-Aldrich).

[0129] Various p38 inhibitors are known in the art. In some embodiments, the inhibitor that directly or indirectly negatively regulates p38 signaling is selected from the group consisting of SB-202190, SB-203580, VX-702, VX-745, PD-169316, RO-4402257 and BIRB-796.

[0130] In one embodiment, a p38 inhibitor according to the present invention binds to its target and reduces the activity of the target by more than 10%, more than 30%, more than 60%, more than 80%, more than 90%, more than 95%, or more than 99% compared to a control as assessed by a cellular assay. Examples of cellular assays for measuring target inhibition are well known in the art as described above.

[0131] SB-203580 may be added to the growth medium at a concentration of 50 nM to 100 μM, or 100 nM to 50 μM, or 1 μM to 50 μM. For example, SB-203580 may be added to the growth medium at approximately 10 μM.

[0132] TGF-beta inhibitors In some embodiments, the proliferation or differentiation medium further comprises a TGF-beta inhibitor.

[0133] TGF-beta signaling is involved in many cellular functions, including cell proliferation, cell fate, and apoptosis. Signaling typically begins with the binding of TGF-beta superfamily ligands to type II receptors, which recruit and phosphorylate type I receptors. Type I receptors then phosphorylate SMADs. SMADs act as transcription factors in the nucleus to control the expression of target genes.

[0134] The TGF-beta inhibitor signaling pathway has previously been implicated in promoting differentiation of progenitor cells. For example, the addition of TGF-beta to liver explants promotes bile duct differentiation in vitro (Clotman et al. (2005) Genes Dev. 19 (16): 1849-54). In addition, the inclusion of TGF-beta inhibitors in the differentiation medium has been shown to inhibit bile duct cell fate and direct cell differentiation more toward a hepatocyte phenotype (see WO2012 / 168930). In particular, the inclusion of TGF-beta inhibitors (such as A83-01) in the differentiation medium has been found to enhance the expression of mature hepatocyte markers and increase the number of hepatocyte-like cells.

[0135] TGF-beta superfamily ligands include bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs), anti-Mullerian hormone (AMH), activin, nodal and TGF-beta. In general, Smad2 and Smad3 are phosphorylated by ALK4, 5 and 7 receptors of the TGF-beta / activin pathway. In contrast, Smad1, Smad5 and Smad8 are phosphorylated as part of the bone morphogenetic protein (BMP) pathway. Although there is some crossover between the pathways, in the context of the present invention, a "TGF-beta inhibitor" or "TGF-beta signaling inhibitor" is preferably an inhibitor of the TGF-beta pathway that acts through Smad2 and Smad3 and / or through ALK4, ALK5 or ALK7. Thus, in some embodiments, the TGF-beta inhibitor is not a BMP inhibitor. That is, the TGF-beta inhibitor is not Noggin. In some embodiments, in addition to the TGF-beta inhibitor, a BMP inhibitor is added to the culture medium. Thus, a TGF-beta inhibitor can be any agent that reduces the activity of the TGF-beta signaling pathway, preferably a signaling pathway that acts through Smad2 and / or Smad3, more preferably a signaling pathway that acts through ALK4, ALK5 or ALK7.

[0136] There are many methods of disrupting the TGF-beta signaling pathway that are known in the art and can be used in conjunction with the present invention. For example, TGF-beta signaling can be disrupted by inhibiting TGF-beta expression with small interfering RNA strategies, inhibiting furin (a TGF-beta activating protease), inhibiting the pathway with physiological inhibitors, neutralizing TGF-beta with monoclonal antibodies, inhibiting TGF-beta receptor kinase 1 (also known as activin receptor-like kinase, ALK5), ALK4, ALK6, ALK7 or other TGF-beta related receptor kinases with small molecule inhibitors, inhibiting Smad 2 and Smad 3 signaling (e.g., by overexpressing their physiological inhibitor Smad 7 or by using thioredoxin as a Smad anchor to disable activation of Smads) (Fuchs, O. Inhibition of TGF-Signalling for the Treatment of Tumor Metastasis and Fibrotic Diseases. Current Signal Transduction Therapy, Volume 6, Number 1, January 2011, pp. 29-43(15)).

[0137] There are various methods known for determining whether a substance is a TGF-beta inhibitor, and they can be used in combination with the present invention.For example, a cellular assay can be used, in which a reporter construct containing the human PAI-1 promoter or Smad binding site and driving a luciferase reporter gene is stably transfected into cells.The inhibition rate of luciferase activity compared to a control group can be used as a measure of compound activity (De Gouville et al. (2005) Br J Pharmacol. 145 (2): 166-177).Therefore, novel TGF-beta inhibitors can be easily identified by those skilled in the art.

[0138] The TGF-beta inhibitor according to the present invention can be a protein, peptide, small molecule, small interfering RNA, antisense oligonucleotide, aptamer or antibody. The inhibitor can be naturally occurring or synthetic. In one embodiment, the TGF-beta inhibitor is an inhibitor of ALK4, ALK5 and / or ALK7. For example, the TGF-beta inhibitor can bind to and directly inhibit ALK4, ALK5 and / or ALK7. Examples of preferred small molecule TGF-beta inhibitors that can be used in the context of the present invention include, but are not limited to, the small molecule inhibitors listed in Table 2 below.

[0139] Table 2. Small molecule TGF-beta inhibitors targeting receptor kinases TIFF2024525079000008.tif194152

[0140] In some embodiments, the TGF-beta inhibitor is optionally a small molecule inhibitor selected from the group consisting of A83-01, SB-431542, SB-505124, SB-525334, LY 364947, SD-208 and SJN 2511.

[0141] In some embodiments, not more than one TGF-beta inhibitor is present in the growth or differentiation medium. In other embodiments, more than one, for example, two, three, four or more TGF-beta inhibitors are present in the growth or differentiation medium. In some embodiments, the growth or differentiation medium of the present invention comprises any one or more of the inhibitors listed in Table 2. The growth or differentiation medium may comprise any combination of one inhibitor listed with another inhibitor. For example, the medium may comprise SB-525334 or SD-208 or A83-01, or may comprise SD-208 and A83-01. Those skilled in the art will recognize that there are many other small molecule inhibitors that are primarily designed to target other kinases, but that may also inhibit TGF-beta receptor kinase at high concentrations. For example, SB-203580 is a p38 MAP kinase inhibitor, but is believed to inhibit ALK5 at high concentrations (e.g., approximately 10 μM or higher). Any such inhibitor that inhibits the TGF-beta signaling pathway may be used in the context of the present invention.

[0142] In some embodiments, the TGF-beta inhibitor (e.g., A83-01) is present in the proliferation or differentiation medium at at least 1 nM, e.g., at least 5 nM, at least 50 nM, at least 100 nM, at least 300 nM, at least 450 nM, or at least 475 nM. For example, the TGF-beta inhibitor (e.g., A83-01) is present in the proliferation or differentiation medium at 1 nM to 200 μM, 10 nM to 200 μM, 100 nM to 200 μM, 1 μM to 200 μM, 10 nM to 100 μM, 50 nM to 100 μM, 50 nM to 10 μM, 100 nM to 1 μM, 200 nM to 800 nM, 350 to 650 nM, or about 500 nM. Thus, in some embodiments, the proliferation or differentiation medium comprises A83-01 at a concentration of about 500 nM.

[0143] Gastrin In some embodiments, the proliferation medium or differentiation medium of the present invention further comprises gastrin. In some embodiments, the differentiation medium of the present invention comprises gastrin at a concentration of 0.01 to 500 nM, 0.1 to 100 nM, 1 to 100 nM, 1 to 20 nM, or 5 to 15 nM. For example, in some embodiments, the proliferation medium or differentiation medium of the present invention comprises gastrin at a concentration of about 5 nM.

[0144] supplement The proliferation and differentiation media of the present invention are preferably supplemented with one or more (e.g., one, two, three, or all) of the compounds selected from the group consisting of B27, N-acetylcysteine, and N2. Thus, in some embodiments, the media further comprises one or more components selected from the group consisting of B27, N2, and N-acetylcysteine. For example, in some embodiments, the media further comprises B27, N-acetylcysteine, and N2. In a preferred embodiment, the media further comprises B27 and N-acetylcysteine.

[0145] B27 (Invitrogen), N-acetylcysteine ​​(Sigma) and N2 (Invitrogen), and nicotinamide (Sigma) are believed to regulate cell growth and support DNA stability.

[0146] In some embodiments, N-acetylcysteine ​​is present in the differentiation medium at a concentration of 0.1-200 mM, 0.1-100 mM, 0.1-50 mM, 0.1-10 mM, 0.1-5 mM, 0.5-200 mM, 0.5-100 mM, 0.5-50 mM, 0.5-10 mM, 0.5-5 mM, 1-100 mM, 1-50 mM, 1-10 mM, 1-5 mM. In some embodiments, N-acetylcysteine ​​is present in the differentiation medium at a concentration of about 1.25 mM.

[0147] In some embodiments, the B27 supplement is "B27 Supplement minus Vitamin A" (also referred to herein as "B27 without Vitamin A" or "B27 wo VitA" and available from Invitrogen, Carlsbad, CA, www.invitrogen.com, now catalog number 12587010, and from PAA Laboratories GmbH, Pasching, Austria, www.paa.com, catalog number F01-002, Brewer et al. (1993) J Neurosci Res. 35(5):567-76). In some embodiments, the B27 supplement can be replaced with a generic formulation that includes one or more of the components selected from the list: biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinyl acetate, sodium selenite, triiodothyronine (T3), DL-alpha tocopherol (vitamin E), albumin, insulin, and transferrin.

[0148] B27 Supplement from PAA Laboratories GmbH is supplied as a 50x concentrate that contains, among other ingredients, biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinol, retinyl acetate, sodium selenite, triiodothyronine (T3), DL-alpha tocopherol (vitamin E), albumin, insulin, and transferrin. Of these ingredients, at least linoleic acid, retinol, retinyl acetate, and triiodothyronine (T3) are nuclear hormone receptor agonists. B27 Supplement may be added to differentiation medium as a concentrate or may be diluted and then added to differentiation medium. B27 Supplement may be used at a final concentration of 1x or other final concentrations (e.g., 0.1x to 4x, 0.1x to 2x, 0.5x to 2x, 1x to 4x, or 1x to 2x). Using B27 Supplement is a convenient way to incorporate biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinol, retinyl acetate, sodium selenite, triiodothyronine (T3), DL-alpha tocopherol (vitamin E), albumin, insulin and transferrin into the differentiation medium of the present invention.It is also envisioned that instead of using B27 Supplement, some or all of these components can be added separately to the differentiation medium.Thus, the differentiation medium can include some or all of these components.

[0149] In some embodiments, retinoic acid is absent from the B27 Supplement used in the differentiation medium and / or is absent from the differentiation medium.

[0150] "N2 Supplement" (also referred to herein as "N2") is available from Invitrogen, Carlsbad, CA, www.invitrogen.com, catalog number 17502-048, and from PAA Laboratories GmbH, Pasching, Austria, www.paa.com, catalog number F005-004, Bottenstein & Sato, PNAS, 76(1):514-517, 1979. N2 Supplement from PAA Laboratories GmbH is supplied as a 100x concentrate containing 500 μg / ml human transferrin, 500 μg / ml bovine insulin, 0.63 μg / ml progesterone, 1611 μg / ml putrescine, and 0.52 μg / ml sodium selenite. N2 Supplement may be added to differentiation medium as a concentrate or may be diluted and then added to differentiation medium. N2 Supplement may be used at a final concentration of 1x or other final concentrations (e.g., 0.1x to 4x, 0.1x to 2x, 0.5x to 2x, 1x to 4x, or 1x to 2x). Using N2 Supplement is a convenient way to incorporate transferrin, insulin, progesterone, putrescine, and sodium selenite into the differentiation medium of the present invention. It is also naturally envisioned that some or all of these components may be added separately to the differentiation medium instead of using N2 Supplement. Thus, the differentiation medium may include some or all of these components.

[0151] In some embodiments where the medium comprises B27, the medium does not further comprise N2. Thus, embodiments of the invention may be adapted to exclude N2, if desired, when B27 is present. In some embodiments, N2 is not present in the medium. In some embodiments where the medium comprises N2, the medium does not further comprise B27. Thus, embodiments of the invention may be adapted to exclude B27, if desired, when N2 is present. In some embodiments, B27 is not present in the medium. In some embodiments, the proliferation or differentiation medium is supplemented with B27 and / or N2.

[0152] In some embodiments, the basal medium is supplemented with 1-3 mM N-acetylcysteine, preferably, the basal medium is supplemented with about 1.25 mM N-acetylcysteine.

[0153] Any suitable pH may be used. For example, the pH of the medium may be in the range of about 7.0 to 7.8, about 7.2 to 7.6, or about 7.4. The pH may be maintained using a buffer. A suitable buffer may be readily selected by one of skill in the art. Buffers that may be used include carbonate buffers (e.g., NaHCO3) and phosphate buffers (e.g., NaH2PO4). These buffers are generally used at about 50 to about 500 mg / l. Other buffers, such as N-[2-hydroxyethyl]-piperazine-N'-[2-ethanesulfonic acid] (HEPES) and 3-[N-morpholino]-propanesulfonic acid (MOPS), may also be used, typically at about 1000 to about 10,000 mg / l. In some embodiments, the buffer is selected from one or more of the list: phosphate buffer (e.g., KH2PO4, K2HPO4, Na2HPO4, NaCl, NaH2PO4), acetate buffer (e.g., HOAc or NaOAc), citrate buffer (e.g., citric acid or Na-citrate), or TRIS buffer (e.g., TRIS, TRIS-HCl) or an organic buffer. In some embodiments, the organic buffer is a zwitterionic buffer such as a Good's buffer, e.g., selected from HEPES, MOPS, MES, ADA, PIPES, ACES, MOPSO, cholamine chloride, BES, TES, DIPSO, acetamidoglycine, TAPSO, POPSO, HEPPSO, HEPPS, tricine, glycineamide, bicine, TAPS, AMPSO, CABS, CHES, CAPS, and CAPSO. A preferred buffer is HEPES, for example, at a concentration of 0.1 to 100 mM, 0.1 to 50 mM, 0.5 to 50 mM, 1 to 50 mM, 1 to 20 mM, or 5 to 15 mM. In some embodiments, HEPES is added to the culture medium at about 10 mM. The differentiation medium may also contain a pH indicator such as phenol red (e.g., about 5 to about 50 mg / liter) to allow easy monitoring of the pH status of the medium.

[0154] The growth medium or differentiation medium used in the present invention may contain one or more amino acids. Those skilled in the art will understand the appropriate type and amount of amino acids to use in differentiation medium. Amino acids that may be present include L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and combinations thereof. Some differentiation medium will contain all of these amino acids. Generally, each amino acid, if present, is present in the medium at about 0.001 to about 1 g / L (usually about 0.01 to about 0.15 g / L), except for L-glutamine, which is present at about 0.05 to about 1 g / L (usually about 0.1 to about 0.75 g / L). The amino acids may be of synthetic origin.

[0155] The growth or differentiation medium used in the present invention may contain one or more vitamins. Those skilled in the art will understand the appropriate type and amount of vitamins to use in the differentiation medium. Vitamins that may be present include thiamine (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), calcium D-pantothenate (vitamin B5), pyridoxal / pyridoxamine / pyridoxine (vitamin B6), folic acid (vitamin B9), cyanocobalamin (vitamin B12), ascorbic acid (vitamin C), calciferol (vitamin D2), DL-alpha tocopherol (vitamin E), biotin (vitamin H) and menadione (vitamin K).

[0156] The growth or differentiation medium used in the present invention may contain one or more inorganic salts. Those skilled in the art will understand the appropriate types and amounts of inorganic salts to use in differentiation medium. Inorganic salts are typically included in differentiation medium to help maintain the osmotic balance of the cells and aid in regulating the membrane potential. Inorganic salts that may be present include calcium, copper, iron, magnesium, potassium, sodium, zinc salts. Salts are usually used in the form of chlorides, phosphates, sulfates, nitrates, and bicarbonates. Particular salts that may be used include CaCl2, CuSO4-5H2O, Fe(NO3)-9H2O, FeSO4-7H2O, MgCl, MgSO4, KCl, NaHCO3, NaCl, Na2HPO4, Na2HPO4-H2O, and ZnSO4-7H2O.

[0157] The osmolarity of the medium can be in the range of about 200 to about 400 mOsm / kg, in the range of about 290 to about 350 mOsm / kg, or in the range of about 280 to about 310 mOsm / kg. The osmolarity of the medium can be less than about 300 mOsm / kg (e.g., about 280 mOsm / kg).

[0158] The growth or differentiation medium used in the present invention may include a carbon energy source in the form of one or more sugars. Those skilled in the art will understand the appropriate types and amounts of sugars to use in the differentiation medium. Sugars that may be present include glucose, galactose, maltose and fructose. The sugar is preferably glucose, particularly D-glucose (dextrose). The carbon energy source will typically be present at about 1 to about 10 g / L.

[0159] The proliferation medium or differentiation medium of the present invention may contain serum. Serum obtained from any suitable source may be used, including fetal bovine serum (FBS), goat serum or human serum. Preferably, human serum is used. Serum may be used at about 1% to about 30% of the medium volume according to conventional techniques.

[0160] In another embodiment, the proliferation medium or differentiation medium of the present invention may contain a serum replacement. A variety of different serum replacement preparations are commercially available and known to those skilled in the art. When a serum replacement is used, the serum replacement may be used at about 1% to about 30% of the medium volume according to conventional techniques.

[0161] In other embodiments, the proliferation or differentiation medium of the present invention may be serum and / or serum replacement-free. A serum-free medium is a medium that does not contain any type of animal serum. A serum-free medium may be preferred to avoid possible foreign body contamination of stem cells. A serum replacement-free medium is a medium that is not supplemented with any commercially available serum replacement preparation.

[0162] In preferred embodiments, the proliferation or differentiation medium is supplemented with purified, natural, semi-synthetic and / or synthetic growth factors and does not contain undefined components such as fetal bovine serum or fetal bovine serum. For example, supplements such as B27 (Invitrogen), N-acetylcysteine ​​(Sigma) and N2 (Invitrogen) stimulate the proliferation of some cells. In some embodiments, the differentiation medium is supplemented with one or more of these supplements, for example, one, any two or all three of these supplements.

[0163] The proliferation or differentiation medium used in the present invention may contain one or more trace elements, such as ions of barium, bromine, cobalt, iodine, manganese, chromium, copper, nickel, selenium, vanadium, titanium, germanium, molybdenum, silicon, iron, fluorine, silver, rubidium, tin, zirconium, cadmium, zinc and / or aluminum.

[0164] The medium may contain a reducing agent, such as beta-mercaptoethanol, at a concentration of about 0.1 mM.

[0165] The proliferation or differentiation medium of the present invention may contain one or more additional agents, such as nutrients or growth factors that have been previously reported to improve stem cell culture, such as cholesterol / transferrin / albumin / insulin / progesterone, putrescine, selenite / other factors.

[0166] Exemplary Differentiation Media Exemplary differentiation media suitable for use in the present invention are summarized in Table 3. These differentiation media are particularly suitable for use in the monolayer membrane derived from intestinal organoid. Differentiation media can be selected to promote the existence or enrichment of specific cell types in the monolayer membrane (for example, one or more cell types listed in Table 3).

[0167] Table 3. Exemplary differentiation media for intestinal organoid-derived monolayers TIFF2024525079000009.tif82152W: Wnt3a or NGS-Wnt, E: EGF, N: Noggin, R: Rspondin3, i: Inhibition of the pathway involved

[0168] The present inventors have found that when organoid-derived monolayer is cultured in cDM (also referred to herein as cCDM), it has higher TEER value and more heterogeneous cell composition than when it is cultured in other differentiation medium.Therefore, in a preferred embodiment, the differentiation medium comprises Notch inhibitor, EGFR pathway inhibitor and Wnt agonist.

[0169] In some embodiments, the differentiation medium comprises a gamma secretase inhibitor (e.g., DAPT, dibenzazepine (DBZ), benzodiazepine (BZ) or LY-411575), a RAS-RAF-MAPK pathway inhibitor (e.g., a MEK inhibitor such as PD0325901), and one or more Wnt agonists selected from the group consisting of Rspondin, Wnt conditioned medium, and Wnt substitute.

[0170] In a preferred embodiment, the differentiation medium comprises DAPT (e.g., at a concentration of about 10 μM), PD0325901 (e.g., at a concentration of about 100 nM), Wnt conditioned medium (e.g., at about 10% of the final volume), and Rspondin (e.g., at a concentration of about 250 ng / mL). In another preferred embodiment, the differentiation medium comprises DAPT (e.g., at a concentration of about 10 μM), PD0325901 (e.g., at a concentration of about 100 nM), Wnt substitute (e.g., NGS-Wnt at a concentration of about 0.1 nM), and Rspondin (e.g., at a concentration of about 250 ng / mL).

[0171] In some embodiments, the differentiation medium comprises a Wnt agonist and a Wnt secretion inhibitor. In some embodiments, the differentiation medium comprises a Rspondin and a Porc inhibitor (e.g., IWP 2, LGK974, or IWP 1). For example, in some embodiments, the differentiation medium comprises a Rspondin (e.g., at a concentration of about 250 ng / mL) and an IWP 2 (e.g., at a concentration of about 1.5 μM).

[0172] In some embodiments, the differentiation medium comprises a Notch inhibitor and a Wnt inhibitor. In some embodiments, the differentiation medium comprises a gamma secretase inhibitor (e.g., DAPT, dibenzazepine (DBZ), benzodiazepine (BZ) or LY-411575) and a Wnt secretion inhibitor such as a Porc inhibitor (e.g., IWP 2, LGK974 or IWP 1). For example, in some embodiments, the differentiation medium comprises DAPT (e.g., at a concentration of about 10 μM) and IWP 2 (e.g., at a concentration of about 1.5 μM).

[0173] In some embodiments, the differentiation medium comprises a Wnt agonist and a Notch inhibitor. In some embodiments, the differentiation medium comprises one or more Wnt agonists selected from the group consisting of Rspondin, Wnt conditioned medium, and Wnt substitute, and a gamma secretase inhibitor (e.g., DAPT, dibenzazepine (DBZ), benzodiazepine (BZ), or LY-411575). For example, in some embodiments, the differentiation medium comprises Rspondin (e.g., at a concentration of about 250 ng / mL), Wnt conditioned medium (e.g., at about 50% of the final volume), and DAPT (e.g., at a concentration of about 10 μM). In other embodiments, the medium comprises a medium comprising Rspondin (e.g., at a concentration of about 250 ng / mL), a Wnt substitute (e.g., NGS-Wnt at a concentration of about 0.1 nM), and DAPT (e.g., at a concentration of about 10 μM).

[0174] In some embodiments, the differentiation medium comprises a Wnt inhibitor, a Notch inhibitor, and an EGFR pathway inhibitor. In some embodiments, the differentiation medium comprises a Wnt secretion inhibitor, such as a Porc inhibitor (e.g., IWP 2, LGK974, or IWP 1), a gamma secretase inhibitor (e.g., DAPT, dibenzazepine (DBZ), benzodiazepine (BZ), or LY-411575), and an EGFR pathway inhibitor selected from an EGFR inhibitor, an EGFR and ErbB2 inhibitor, and a RAS-RAF-MAPK pathway inhibitor (e.g., a MEK inhibitor, such as PD0325901). For example, in some embodiments, the medium comprises IWP-2 (e.g., at a concentration of about 1.5 μM), DAPT (e.g., at a concentration of about 10 μM), and PD0325901 (e.g., at a concentration of about 100 nM).

[0175] In any of the above embodiments, the differentiation medium may further comprise a TGF-beta inhibitor, gastrin, a BMP inhibitor and / or a receptor tyrosine kinase ligand. For example, in some embodiments, the differentiation medium further comprises A83-01 (e.g., at a concentration of about 500 nM), gastrin (e.g., at a concentration of about 5 nM), noggin (e.g., at a concentration of about 100 ng / mL) and EGF (e.g., at a concentration of about 50 ng / mL). In other embodiments, the differentiation medium further comprises A83-01 (e.g., at a concentration of about 500 nM), gastrin (e.g., at a concentration of about 5 nM), noggin (e.g., at a concentration of about 100 ng / mL) and EGF (e.g., at a concentration of about 50 ng / mL).

[0176] Preferably, the differentiation medium does not contain p38 MAPK inhibitors (e.g., SB202190) or nicotinamide.

[0177] In some embodiments, especially when organoid is derived from lung, differentiation medium comprises one or more receptor tyrosine kinase, Wnt agonist, Notch inhibitor and BMP pathway activator.In some embodiments, differentiation medium further comprises Rho kinase inhibitor and p38 inhibitor.In a preferred embodiment, differentiation medium comprises (i) FGF (e.g., FGF-7 at about 25ng / mL concentration and FGF-10 at about 100ng / mL concentration), (ii) Rspondin (e.g., Rspondin-3 at about 250ng / mL concentration), (iii) Notch inhibitor (e.g., DAPT at about 10μM concentration), (iv) BMP (e.g., BMP4 at about 10ng / mL concentration), (v) Rho-kinase inhibitor (e.g., Y-27632 at about 5μM concentration), and (vi) p38 kinase inhibitor (e.g., SB202190 at about 500nM concentration). The pulmonary differentiation medium used herein has been previously described by van de Vaart et al. (EMBO reports (2021) 22: e52058).

[0178] The present invention also provides a differentiation medium comprising a Notch inhibitor, an EGFR pathway inhibitor and a Wnt agonist, eg, according to any of the embodiments described herein.

[0179] Culture vessel and extracellular matrix The organoid-derived monolayer of the present invention is cultured on a semi-permeable membrane. Any suitable culture vessel or culture system that includes a semi-permeable membrane can be used, for example, Transwell® 96-well permeable support (Corning®). The culture vessel or culture system is divided into an apical compartment and a basal compartment by a semi-permeable membrane. In some embodiments, culture medium is present in both the apical compartment and the basal compartment. In some embodiments, the apical compartment and the basal compartment contain the same medium, for example, the same growth medium or the same differentiation medium. In other embodiments, the medium in the apical compartment is different from the medium present in the basal compartment.

[0180] In some embodiments, the method of obtaining the organoid-derived monolayer of the present invention comprises removing the growth medium or differentiation medium from apical compartment.This method is known in the art as "air-liquid interface" culture, or ALI culture, and is particularly suitable for the culture of pulmonary organoid-derived monolayer, as shown in the examples.

[0181] The medium may be refreshed; that is, the medium is removed and replenished as needed. In some embodiments, the medium is refreshed every 1, 2, 3, 4, 5, 6 or 7 days. Preferably, the medium in both the apical and basal compartments is refreshed every 2-3 days. Preferably, the medium is first removed from the basal compartment, followed by the medium from the apical compartment. Then, medium is added to the apical compartment, followed by the medium to the basal compartment. When a component is "added" to or "removed" from the medium, in some embodiments, this may mean that the medium itself is removed from the apical and / or basal compartments, and that new medium containing the "added" component or excluding the "removed" component is placed in the apical and / or basal compartments.

[0182] In some embodiments, the organoid-derived monolayer of the present invention is cultured in contact with extracellular matrix (ECM).For example, the semipermeable membrane can be coated with ECM.In some embodiments, the semipermeable membrane is coated with ECM by adding ECM to the apical compartment and incubating the membrane for a period of time (for example, about 30 minutes, about 1 hour, about 2 hours or more), and then the suspension of organoid-derived single cells and / or organoid fragments is seeded.In some embodiments, particularly when the ECM is Matrigel™, the semipermeable membrane is coated with ECM at a concentration of about 2.5% for about 1 hour.

[0183] Any suitable ECM can be used.The organoid-derived monolayer is preferably cultured in a microenvironment that at least partially mimics the cell niche where the cells that compose the membrane are naturally present.The cell niche is partially determined by the cells and by the ECM that the cells secrete in said niche.The cell niche can be mimicked by culturing the organoid-derived monolayer in the presence of a biomaterial or synthetic material that allows interaction with cell membrane proteins such as integrins.Thus, the ECM described herein is any biomaterial or synthetic material or combination thereof that mimics the cell niche in vivo by interacting with cell membrane proteins such as integrins.

[0184] In the preferred method of the present invention, organoid-derived monolayer is cultured in contact with ECM."In contact" means physical or mechanical or chemical contact, which means that force must be used to separate said organoid-derived monolayer from said extracellular matrix.In some embodiments, organoid-derived monolayer is attached to ECM.The culture solution of the present invention can be diffused into three-dimensional ECM.

[0185] One type of ECM is that secreted by epithelial cells, endothelial cells, parietal endoderm-like cells (e.g., Englebreth Holm Swarm Parietal Endoderm-Like cells, described in Hayashi et al. (2004) Matrix Biology 23:47-62) and connective tissue cells. This ECM is composed of various polysaccharides, water, elastin, and glycoproteins, where glycoproteins include collagen, entactin (nidogen), fibronectin, and laminin. Thus, in some embodiments, the ECM for use in the methods of the invention comprises one or more components selected from the list: polysaccharides, elastin, and glycoproteins, e.g., glycoproteins include collagen, entactin (nidogen), fibronectin, and / or laminin. For example, in some embodiments, collagen is used as the ECM. Various types of ECM are known, composed of various compositions including various types of glycoproteins and / or combinations of different glycoproteins.

[0186] ECM can be produced by, for example, culturing ECM-producing cells, such as epithelial cells, endothelial cells, parietal endoderm-like cells or fibroblasts, in a container, then removing these cells and adding a suspension of cells and / or organoid fragments obtained by digesting or dissociating one or more organoids. Examples of extracellular matrix-producing cells include chondrocytes, which mainly produce collagen and proteoglycans, fibroblasts, which mainly produce type IV collagen, laminin, interstitial procollagen, and fibronectin, and colon myofibroblasts, which mainly produce collagen (types I, III, and V), chondroitin sulfate proteoglycans, hyaluronic acid, fibronectin, and tenascin-C. These are "naturally produced ECMs". Naturally produced ECMs can be commercially provided. Examples of commercially available extracellular matrices include extracellular matrix proteins (Invitrogen) and basement membrane preparations derived from mouse Engelbreth-Holm-Swarm (EHS) sarcoma cells (e.g., Cultrex® Basement Membrane Extract (Trevigen, Inc.) or Matrigel™ (BD Biosciences)).

[0187] Thus, in some embodiments, the ECM is a naturally produced ECM. In some embodiments, the ECM is a laminin-containing ECM, such as Matrigel™ (BD Biosciences). In some embodiments, the ECM is Matrigel™ (BD Biosciences), which contains laminin, entactin, and collagen IV. In some embodiments, the ECM contains laminin, entactin, collagen IV, and heparin sulphate proteoglycan (e.g., Cultrex® Basement Membrane Extract Type 2 (Trevigen, Inc.)). In some embodiments, the ECM contains at least one glycoprotein, such as collagen and / or laminin. A preferred ECM for use in the methods of the invention contains collagen and laminin. A more preferred ECM contains laminin, entactin, and collagen IV. If desired, a mixture of naturally produced or synthetic ECM materials may be used.

[0188] In another embodiment, the ECM can be a synthetic ECM. For example, a synthetic ECM such as ProNectin (Sigma Z378666) can be used. In a further example, the ECM can be a plastic (e.g., polyester, or hydrogel). In some embodiments, the synthetic matrix can be coated with a biomaterial (e.g., one or more glycoproteins such as collagen or laminin).

[0189] In some embodiments, the growth medium or differentiation medium further comprises an integrin agonist (e.g., as described in WO2020 / 234250). Specific examples of integrin agonists include anti-integrin antibodies, such as anti-b1 integrin antibodies (e.g., TS2 / 16, 12G10, 8A2, 15 / 7, HUTS-4, 8E3, N29 and 9EG7 antibodies). Integrin agonists can be used in place of or in addition to extracellular matrix.

[0190] co-culture In some embodiments, the monolayer contains epithelial cells co-cultured with non-epithelial cells. In other embodiments, the monolayer contains only epithelial cell types. Methods for co-culturing organoids with immune cells are described in WO2019 / 122388. Co-culturing organoid-derived monolayers with immune cells can be useful for studying the physiology of disease and / or the suitability (efficacy and / or safety) of candidate drugs for disease treatment. Thus, in some embodiments, the organoid-derived monolayers of the present invention are co-cultured with immune cells.

[0191] Properties of the organoid-derived monolayer of the present invention In some embodiments, the TEER of the organoid-derived monolayers of the present invention is about 10, about 50, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400, or about 1500 Ω·cm 2 In some embodiments, the TEER of the organoid-derived monolayer of the present invention is about 2, about 5, about 10, about 50, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400, or about 1500 Ω·cm 2 It is.

[0192] In some embodiments, the TEER of the organoid-derived monolayer of the present invention is about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400, or about 1500 Ω·cm 2 Preferably, the TEER of the organoid-derived monolayer of the present invention is 100 Ω cm 2 It's super.

[0193] In some embodiments, particularly when the organoid-derived monolayer is derived from the kidney, the TEER of the monolayer is greater than or equal to 25 Ω cm 2 Super, 50Ω cm2 Super, 75Ω cm 2 Super, 100Ω cm 2 Super, 200Ω cm 2 Super, 300Ω cm 2 Super, 400Ω cm 2 Super, 500Ω cm 2 Super, 600Ω cm 2 Super, 700Ω cm 2 Super, 800Ω cm 2 Super, 900Ω cm 2 Super, 1000Ω cm 2 Super, 1100Ω cm 2 Super, 1200Ω cm 2 Super, 1300Ω cm 2 Over 1400 Ω cm 2 It's super.

[0194] In some embodiments, the organoid-derived monolayers of the present invention, derived, for example, from intestinal organoids, contain the following cell types: Lgr5 + The cellular composition of the organoid-derived monolayer may be evaluated by detecting or quantifying the expression of one or more marker genes. Lgr5 is a marker for Lgr5. + A stem cell marker. Ki67 is a marker for Lgr5 + It is a marker for proliferative cells such as stem cells. Goblet cells can be detected by performing mucus staining, e.g., Alcian Blue staining, or by detecting expression of Mucin-2 (Muc2), as described herein. Intestinal alkaline phosphatase (ALPI or ALPI1) is a marker for intestinal absorptive epithelial cells. Lysozyme is a marker for Paneth cells. Chromogranin A is a marker for enteroendocrine cells.

[0195] In some embodiments, for example, the organoid-derived monolayer of the present invention derived from lung organoid comprises one or more (preferably all) of the following cell types: club cell, basal cell, ciliated cell, goblet cell, type I pneumocyte and type II pneumocyte.In some embodiments, for example, the organoid-derived monolayer of the present invention derived from lung organoid expresses one or more (preferably all) of the following genes that are markers of specific cell types: KRT5 (lung basal cell marker), SPDEF (goblet cell marker), FOXJ1 (ciliated cell marker), and SFTPA1 (alveolar marker, particularly type II pneumocyte).

[0196] In some embodiments, for example, the organoid-derived monolayer of the present invention derived from kidney organoid comprises one or more (preferably all) of the following cell types: proximal tubule cell, renal epithelial cell, Henle's loop cell, distal tubule cell and collecting duct cell.ABCC4 is proximal tubule marker, PAX8 is renal epithelial marker, CLDN10 is Henle's loop marker, SLC12A3 is distal tubule marker, and AQP3 is collecting duct marker.

[0197] Depending on the identity of the marker, expression of the marker may be assessed by RT-PCR, immunohistochemistry or tissue staining after 3, 4, 5, 6, 7, 8, 9 or more days of culture in proliferation or differentiation medium as described herein. In some embodiments, expression of the marker is measured after 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more days (e.g., 16 days) of culture in proliferation and / or differentiation medium as described herein.

[0198] The term "expressed" is used to describe the presence of a marker in a cell. To be considered expressed, a marker must be present at a detectable level. By "detectable level" is meant that the marker can be detected using one of the standard testing methods, such as PCR, blotting, or FACS analysis. A gene is considered to be expressed by the cell population of the present invention if expression can be reasonably detected after 30 PCR cycles (corresponding to an intracellular expression level of at least about 100 copies per cell). The terms "express" and "expression" have the same meaning. Below this threshold, an expression level is considered to be unexpressed. A comparison of the expression level of a marker in a cell of the present invention with the expression level of the same marker in another cell, such as an embryonic stem cell, can be preferably performed by comparing two cell types isolated from the same species. Preferably, the species is mammalian, and more preferably, the species is human. Such a comparison can be conveniently performed using reverse transcription polymerase chain reaction (RT-PCR) experiments.

[0199] In some embodiments, the organoid-derived monolayer of the present invention expresses one or more of the following markers: ALPI, MUC2, lysozyme, Ki67 and Lgr5.In some embodiments, the organoid-derived monolayer of the present invention expresses Lgr5 and Muc2.In some embodiments, the organoid-derived monolayer of the present invention does not express ALPI.In some embodiments, the organoid-derived monolayer of the present invention expresses Lgr5 and Muc2, and does not express ALPI.In some embodiments, the organoid-derived monolayer of the present invention expresses lysozyme.

[0200] In some embodiments, especially when the monolayer is derived from kidney organoid, the organoid-derived monolayer of the present invention expresses one or more, preferably all, of ABCC4, PAX8, CLDN10, SLC12A3, AQP3, OCT2, MATE1 and MATE2-K.In some embodiments, the organoid-derived monolayer expresses one or more, preferably all, of PAX8, CLDN10, AQP3, OCT2, MATE1 and MATE2-K.In some embodiments, the organoid-derived monolayer expresses one or more, preferably all, of PAX8, OCT2, MATE1 and MATE2-K.In some embodiments, the organoid-derived monolayer does not express OAT1 or OAT3.

[0201] In some embodiments, especially when the monolayer is derived from lung organoid, the organoid-derived monolayer of the present invention expresses one or more, preferably all, of KRT5, SPDEF, FOXJ1 and SFTPA1.In some embodiments, the organoid-derived monolayer expresses one or more, preferably all, of KRT5, SPDEF and FOXJ1.In some embodiments, the organoid-derived monolayer expresses one or more, preferably all, of KRT5, SPDEF and SFTPA1.

[0202] In some embodiments, the organoid-derived monolayer of the present invention has polarity along the apical-basal axis.In some embodiments, when an EGFR inhibitor such as gefitinib is applied to the basal side of the monolayer, the TEER of the organoid-derived monolayer of the present invention is decreased, and / or the permeability of the organoid-derived monolayer of the present invention to dyes such as Lucifer Yellow is increased.In some embodiments, when an EGFR inhibitor such as gefitinib is applied to the apical side of the monolayer, the TEER of the organoid-derived monolayer of the present invention is not decreased, and / or the permeability of the organoid-derived monolayer of the present invention to dyes such as Lucifer Yellow is not increased.

[0203] In some embodiments, the organoid-derived monolayer of the present invention is impermeable to dyes such as Lucifer Yellow.In some embodiments, the organoid-derived monolayer of the present invention becomes permeable to dyes such as Lucifer Yellow when it is injured, for example, by the tip of a pipette.

[0204] In some embodiments, the organoid-derived monolayer of the present invention has a smooth apical surface.In some embodiments, the organoid-derived monolayer of the present invention has an invaginated apical morphology.In some embodiments, the organoid-derived monolayer of the present invention has ciliated apical surface (see, for example, the lung organoid-derived monolayer of Figure 16C).In some embodiments, particularly when the monolayer is derived from lung organoid, the monolayer has a bubble-like structure.

[0205] The organoid-derived monolayer may be pseudostratified (see, for example, lung organoid-derived monolayers in Figures 16A and 16C).

[0206] In some embodiments, the organoid-derived monolayer of the present invention has transport function.That is, it can transport substrate from apical compartment to basal compartment or from basal compartment to apical compartment.Transport function can be measured using the assay described herein.

[0207] Uses of organoid-derived monolayers Similarly, the use of organoid-derived monolayer membrane described herein is provided.For example, the present invention provides the use of organoid-derived monolayer membrane in drug discovery screening, in toxicity assay, in histogenesis, cell lineage and differentiation pathway research, in identifying the chemical and / or neuronal signals that lead to the release of each hormone, in gene expression research, including recombinant gene expression, in the research of the mechanism involved in tissue injury and tissue repair, in the research of inflammatory and infectious diseases, in the research of pathogenic mechanism, or in the research of cell transformation mechanism and the pathogenesis of cancer.

[0208] The present invention provides the use of the organoid-derived monolayers of the present invention in drug screening, (drug) target validation, (drug) target discovery, in toxicology and toxicological screening, in personalized medicine, and / or as ex vivo cell / organ models, such as disease models.

[0209] The organoid-derived monolayer of the present invention is believed to faithfully represent the in vivo state.Therefore, the organoid of the present invention can be used not only to provide a normal ex vivo cell / organ model, but also as an ex vivo disease model.

[0210] The organoid-derived monolayer of the present invention can also be used to culture pathogens, and therefore can be used as ex vivo infection model.The examples of pathogens that can be cultured using the organoid of the present invention include viruses, bacteria, prions or fungi that cause disease in their animal hosts.Therefore, the organoid-derived monolayer of the present invention can be used as disease model that represents infection state.In some embodiments of the present invention, the organoid can be used for vaccine development and / or vaccine production.

[0211] Thus, diseases that can be studied by the organoid-derived monolayers of the present invention include genetic, metabolic, pathogenic, inflammatory diseases, and the like, including, but not limited to, diabetes (such as type I or type II), cystic fibrosis, carcinoma, adenocarcinoma, adenoma, enteropancreatic neuroendocrine tumors, inflammatory bowel disease (such as Crohn's disease or ulcerative colitis), celiac disease, and leaky gut syndrome.

[0212] Traditionally, cell lines and more recently iPS cells have been used as ex vivo cell / organ models and / or disease models (see, for example, Robinton et al. Nature 481, 295, 2012). However, these methods have many challenges and drawbacks. For example, cell lines cannot be obtained from every patient (only certain biopsies succeed in obtaining cell lines), and therefore cell lines cannot be used for personalized diagnosis and personalized medicine. iPS cells usually require some degree of genetic manipulation to reprogram the cells to a specific cell fate. Alternatively, iPS cells must be kept in culture for a minimum time, since they are subjected to culture conditions that affect the integrity of the karyotype (this is also the case for human embryonic stem cells). This means that iPS cells cannot accurately represent the in vivo condition, but instead, they attempt to mimic the behavior of in vivo cells. Cell lines and iPS cells are also genetically unstable.

[0213] In contrast, the organoid-derived monolayer of the present invention provides a genetically stable platform that faithfully represents in vivo conditions.In some embodiments, the organoid-derived monolayer of the present invention comprises all the differentiated cell types present in the corresponding in vivo conditions.In other embodiments, the organoid-derived monolayer of the present invention can be further differentiated to provide all the differentiated cell types present in vivo.Therefore, the organoid-derived monolayer of the present invention can be used to obtain mechanistic insights into various diseases and treatments, to carry out in vitro drug screening, to evaluate potential treatments, to identify candidate targets (e.g., proteins) for the development of new (drug) therapies in the future, and / or to explore gene repair in conjunction with cell replacement therapy.

[0214] For these reasons, the organoid-derived monolayers of the present invention can be tools for drug screening, target validation, target discovery, toxicology and toxicological screening, and personalized medicine.

[0215] Therefore, the present invention also provides the use of the organoid-derived monolayer of the present invention in the assay for evaluating epithelial viability, metabolic activity, permeability, barrier function integrity and / or transport protein activity.Described herein is the method for evaluating the viability, permeability and barrier function integrity of organoid-derived monolayer, and the activity of transport protein in organoid-derived monolayer.

[0216] The present invention also provides a method for identifying a compound capable of modulating epithelial viability, metabolic activity, permeability, barrier function integrity and / or transport protein activity, comprising the steps of: i. contacting an organoid-derived monolayer, e.g., an organoid-derived monolayer as described herein, with one or more candidate molecules; ii. Assessing the viability, metabolic activity, permeability and / or barrier function integrity of the organoid-derived monolayer and / or the activity of transport proteins in the organoid-derived monolayer.

[0217] "Modulating" can mean improving, restoring, damaging or inhibiting epithelial viability, metabolic activity, permeability, barrier function integrity and / or transport protein activity.

[0218] In some embodiments, the one or more candidate molecules are a library of candidate molecules, or part of a library of candidate molecules.

[0219] The present invention also provides a method for assessing the effect of a compound on epithelial viability, metabolic activity, permeability, barrier function integrity and / or transport protein activity, comprising the steps of: i. contacting an organoid-derived monolayer, e.g., an organoid-derived monolayer as described herein, with the compound; and ii. Assessing the viability, metabolic activity, permeability and / or barrier function integrity of the organoid-derived monolayer and / or the activity of transport proteins in the organoid-derived monolayer.

[0220] In some embodiments, the compound is an approved or experimental drug for a disease or disorder of the digestive system, such as, for example, inflammatory bowel disease (e.g., Crohn's disease or ulcerative colitis), celiac disease or leaky gut syndrome, etc. In some embodiments, the compound is tofacitinib.

[0221] The inventors demonstrate that a combination of proinflammatory cytokines can be used to induce epithelial barrier injury in organoid-derived monolayers, which may provide a useful model to study the effects of compounds on epithelial viability, metabolic activity, permeability, barrier integrity and / or transport protein activity.

[0222] Thus, in some embodiments, the method of assessing the effect of a compound on epithelial viability, metabolic activity, permeability, barrier function integrity and / or transport protein activity described herein, or the method of identifying a compound capable of modulating epithelial viability, metabolic activity, permeability, barrier function integrity and / or transport protein activity described herein, further comprises contacting the organoid-derived monolayer with one or more inflammatory cytokines, for example, selected from the group consisting of IL-1α, IL-1β, IL-2, IL-12, IL-17, IL-18, IFN-γ, and TNF-α. Preferably, the one or more inflammatory cytokines are selected from the group consisting of IFN-γ, TNF-α, and IL-1α. In some embodiments, the one or more inflammatory cytokines include IFN-γ, TNF-α, and IL-1α. In some embodiments, the one or more inflammatory cytokines include IFN-γ and TNF-α. In some embodiments, the one or more inflammatory cytokines include TNF-α and IL-1α. The step of contacting organoid-derived monolayer with one or more inflammatory cytokines can be carried out before, after or simultaneously with the step of contacting said monolayer with said compound or one or more candidate molecules.In a preferred embodiment, the step of contacting organoid-derived monolayer with one or more inflammatory cytokines is followed by the step of contacting said monolayer with said compound or one or more candidate molecules.

[0223] The present invention also provides a method for identifying mutations associated with epithelial viability, metabolic activity, permeability, barrier function integrity and / or transport protein activity, comprising the steps of: i. assessing the viability, metabolic activity, permeability and / or barrier function integrity of an organoid-derived monolayer, e.g., an organoid-derived monolayer as described herein, and / or the activity of transport proteins in an organoid-derived monolayer, e.g., an organoid-derived monolayer as described herein; ii. Determining the presence of one or more mutations in the genome of one or more cells in the organoid-derived monolayer.

[0224] Mutations can be identified by sequencing the genome of one or more cells in the organoid-derived monolayer and / or by performing single nucleotide polymorphism (SNP) microarrays on DNA isolated from one or more cells in the organoid-derived monolayer.

[0225] The present invention also provides i. obtaining an organoid-derived monolayer from said human subject using a method described herein; and ii. Testing the viability, metabolic activity, permeability and / or barrier function integrity of the organoid-derived monolayer and / or the activity of transport proteins in the organoid-derived monolayer. wherein a test result above or below a reference value indicates the presence of said disease or affliction, or an increased risk of said disease or affliction, in a human subject, which affects epithelial viability, metabolic activity, permeability, barrier function integrity and / or transport protein activity, comprising:

[0226] In some embodiments, the reference value is a value obtained from a control. In some embodiments, the control is an organoid-derived monolayer obtained from a healthy human subject.

[0227] In some embodiments, the disease or affliction is a disease or disorder of the digestive system. In some embodiments, the disease or affliction is inflammatory bowel disease (e.g., Crohn's disease or ulcerative colitis), celiac disease or leaky gut syndrome. Preferably, the disease or affliction is inflammatory bowel disease (e.g., Crohn's disease or ulcerative colitis).

[0228] The present invention also provides a method for predicting the likelihood that a patient will respond to a candidate compound, comprising the steps of: i. obtaining an organoid-derived monolayer from said patient using a method described herein; ii. contacting the organoid-derived monolayer with the compound; and iii. Assessing the viability, metabolic activity, permeability and / or barrier function integrity of the organoid-derived monolayer and / or the activity of transport proteins in the organoid-derived monolayer.

[0229] In some embodiments, the patient has a disease or disorder of the digestive system. In some embodiments, the disease or disorder is inflammatory bowel disease (e.g., Crohn's disease or ulcerative colitis), celiac disease or leaky gut syndrome. Preferably, the patient has inflammatory bowel disease (e.g., Crohn's disease or ulcerative colitis). In some embodiments, the candidate compound is an approved or experimental drug for any of the diseases or disorders listed above. In some embodiments, the candidate compound is tofacitinib.

[0230] Assessment of viability, metabolic activity, permeability, barrier integrity and / or transport protein activity viability The viability of the organoid-derived monolayers of the present invention can be measured by any suitable method, such as Hoechst staining, propidium iodide staining in FACS, or preferably, for example, using an ATP-based assay as described herein.

[0231] Transepithelial Electrical Resistance (TEER) In some embodiments, the integrity of the barrier function of the organoid-derived monolayer is evaluated by measuring transepithelial electrical resistance (TEER). TEER measurement is a widely accepted method for analyzing the tight junction dynamics and barrier function integrity in physiological barriers of biological models, such as epithelial monolayers. Methods for measuring TEER have been described (see Srinivasan, B. et al. TEER measurement techniques for in vitro barrier model systems. Journal of Laboratory Automation. 20 (2), 107-126 (2015); and Blume, L.-F. et al. Temperature corrected transepithelial electrical resistance (TEER) measurement to quantify rapid changes in paracellular permeability. Die Pharmazie. 65 (1), 19-24 (2010)). TEER can be measured using a manual TEER meter or an automated TEER measurement robot.

[0232] transparency Permeability can be used as an index of monolayer integrity. Permeability can be transcellular or paracellular. Paracellular permeability is controlled by tight junctions.

[0233] In some embodiments, the evaluation of the permeability of the organoid-derived monolayer includes measuring the passive diffusion rate of the reporter compound from the apical side to the basolateral side of the monolayer. Any suitable reporter compound can be used. In some embodiments, the reporter compound is a dye. In some embodiments, the reporter compound is a labeling compound, for example, a radioactive labeling compound, a fluorescent labeling compound, or a dye labeling compound. Preferably, the reporter compound is Lucifer Yellow. In other embodiments, the reporter compound is a dextran, which is optionally labeled with a dye, for example, a fluorescent dye such as tetramethylrhodamine isothiocyanate (TRITC). In some embodiments, the concentration of the reporter compound in the apical and / or basolateral compartment is measured using mass spectrometry. In some embodiments, the concentration of the reporter compound in the apical and / or basolateral compartment is measured using liquid chromatography-mass spectrometry. In some embodiments, the concentration of the reporter compound in the apical and / or basolateral compartment is measured using colorimetry.

[0234] In some embodiments, the passive diffusivity of the reporter compound across the monolayer is measured by applying the reporter compound to the apical compartment and measuring the amount of the reporter compound in the basolateral compartment. In other embodiments, the passive diffusivity of the reporter compound across the monolayer is measured by applying the reporter compound to the basolateral compartment and measuring the amount of the reporter compound in the apical compartment. The amount of the reporter compound in the apical or basolateral compartment can be measured after, for example, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours or more of incubation period. In some embodiments, the amount of the reporter compound in the apical or basolateral compartment is measured repeatedly, for example, every minute or every hour.

[0235] Transport protein activity The transport function of the organoid-derived monolayer of the present invention can be evaluated by evaluating the activity of transport protein in the monolayer. In some embodiments, the activity evaluation of transport protein comprises measuring the transport rate of the substrate of transport protein across the monolayer, optionally in the presence of an inhibitor of said transport protein. In some embodiments, the activity evaluation of transport protein comprises measuring the transport rate of the substrate of transport protein into the cells of the monolayer, optionally in the presence of an inhibitor of said transport protein. In some embodiments, the transport protein is selected from P-glycoprotein 1 (Pgp1, also known as multidrug resistance protein 1 (MDR1) or ATP-binding cassette subfamily B member 1 (ABCB1)), breast cancer resistance protein (BCRP or ABCG2), peptide transporter 1 (PEPT1) and multidrug resistance protein 2 (MRP2). In some embodiments, the transport protein is selected from P-glycoprotein 1 (Pgp1, also known as multidrug resistance protein 1 (MDR1) or ATP-binding cassette subfamily B member 1 (ABCB1)), breast cancer resistance protein (BCRP or ABCG2), peptide transporter 1 (PEPT1), multidrug resistance protein 2 (MRP2), multidrug resistance protein 1 (MRP1, also known as ABCC1), and organic cation transporter 2 (Oct2, also known as SLC22A2). In some embodiments, the substrate is labeled, e.g., with a fluorescent, radioisotope, or dye. In some embodiments, the substrate is a dye. In some embodiments, the concentration of the substrate in the apical and / or basolateral compartment is measured using mass spectrometry. In some embodiments, the concentration of the substrate in the apical and / or basolateral compartment is measured using liquid chromatography mass spectrometry. In some embodiments, the concentration of the substrate in the apical and / or basolateral compartment is measured using a colorimetric method. In some embodiments, the amount of substrate that has been transported into cells of the monolayer (ie, intracellular accumulation) is assessed by measuring intracellular fluorescence.

[0236] Exemplary substrates and inhibitors suitable for use in the present invention, and their target transport proteins, are outlined in Table 4.

[0237] Table 4: Exemplary transport proteins and their substrates and inhibitors TIFF2024525079000010.tif68152

[0238] In a preferred embodiment, the transport protein is Pgp1, the substrate is rhodamine 123, and the inhibitor is PSC833. In another preferred embodiment, the transport protein is Pgp1, the substrate is calcein AM, and the inhibitor is PSC833. In another preferred embodiment, the transport protein is MRP1, the substrate is calcein AM, and the inhibitor is MK571. In another preferred embodiment, the transport protein is OCT2, the substrate is rhodamine 123, and the inhibitor is decinium-22.

[0239] In some embodiments, the activity of more than one, e.g., two, transport proteins is assessed simultaneously. In such embodiments, two or more transport protein inhibitors may be used.

[0240] kit The present invention also provides a kit for generating the organoid-derived monolayer of the present invention, comprising the organoid described herein and one or more culture media.In particular, the kit can comprise the organoid described herein, growth medium, and optionally one or more differentiation media.The kit can further comprise cell dissociation reagent, ROCK inhibitor, extracellular matrix, and one or more semipermeable membranes.In some embodiments, the membrane is provided pre-coated with extracellular matrix.

[0241] The present invention further provides kits comprising a culture medium, such as a growth medium or a differentiation medium, as described herein. In a preferred embodiment, the kit comprises a differentiation medium comprising a Notch inhibitor, an EGFR pathway inhibitor, and a Wnt agonist. Exemplary differentiation media comprising a Notch inhibitor, an EGFR pathway inhibitor, and a Wnt agonist are described herein.

[0242] The present invention further provides a kit for preparing a differentiation medium comprising a Notch inhibitor, an EGFR pathway inhibitor and a Wnt agonist. Suitable Notch inhibitors, EGFR pathway inhibitors and Wnt agonists are described herein. In a preferred embodiment, the kit comprises DAPT, PD0325901, a Wnt conditioned medium and Rspondin. In another preferred embodiment, the kit comprises DAPT, PD0325901, a Wnt surrogate (e.g., NGS-Wnt) and Rspondin.

[0243] The present invention further provides a kit for evaluating the barrier function and transport function of organoid-derived monolayer membrane, comprising one or more of the following components: gefitinib, staurosporine, Lucifer Yellow, calcein AM, rhodamine 123, P-gp inhibitor (e.g., PSC-833), OCT2 inhibitor (e.g., decinium-22), tofacitinib and one or more inflammatory cytokines.One or more of these components can also be provided as part of a kit comprising the above-mentioned organoid and one or more culture media.

[0244] definition As used herein, the verb "comprise" and its conjugations are used in an open-ended sense, meaning that the item preceding the word is included, but not excluding any item not specifically mentioned. In addition, the verb "consisting of" may be replaced with "consisting essentially of" if necessary, meaning that the product defined herein may include additional component(s) other than the specifically identified components, and said additional component(s) do not modify the specific characteristics of the invention. In addition, the method defined herein may include additional step(s) other than the specifically identified steps, and said additional step(s) do not modify the specific characteristics of the invention. In addition, the reference to an element by the indefinite article "a" or "an" does not exclude the possibility that there is more than one element, unless the context clearly requires that there is one element and that there is only one of the elements. Thus, the indefinite article "a" or "an" usually means "at least one".

[0245] As used herein, the term "about" or "approximately" means that the indicated value may vary by + / - 10%. The value may also be read as an exact value, and therefore the term "about" may be omitted. For example, the term "about 100" includes 90 to 110, and also includes 100.

[0246] The term "digestive system" includes the digestive tract as well as the liver, pancreas and gallbladder.

[0247] The term "digestive tract" includes the mouth, esophagus, stomach, intestines and anus.

[0248] The term "intestinal tract" includes the colon and small intestine.

[0249] The term "small intestine" includes the duodenum, jejunum, and ileum.

[0250] The term "lung" includes the trachea, bronchi, bronchioles, alveolar ducts and alveoli.

[0251] The term "kidney" includes the ureter, cortex, medulla, renal pelvis, and calyx.

[0252] All patent and literature references cited herein are hereby incorporated by reference in their entirety.

[0253] The following examples are presented for illustrative purposes only and are not intended to limit the scope of the present invention in any way. [Brief description of the drawings]

[0254] Description of the drawings By way of example, embodiments of the invention will now be described with reference to the following drawings, in which:

[0255] [Figure 1] Figure 1 illustrates the formation of an organoid-derived monolayer after seeding a single cell onto a membrane. (A) A single cell immediately after seeding onto the membrane. On average, (B) the monolayer becomes ~50% confluent 1-3 days after seeding, (C) ~90% confluent after 3-5 days, and (D) a complete monolayer forms around 4-7 days. Scale bar = 100 μm. [Diagram 2] Illustrating the enrichment of specific cell types in organoid-derived monolayers. (A) Monolayer after 8 days in IEM. (B) Monolayer enriched for intestinal absorptive epithelial cells after 4 days in IEM followed by 4 days in eDM. (C) Monolayer enriched for goblet cells and other cell types after 4 days in IEM followed by 4 days in cDM. Scale bar = 100 μm. Abbreviations: IEM = intestinal organoid growth medium, eDM = intestinal absorptive epithelial cell differentiation medium, cDM = combined differentiation medium. [Figure 3A] Figure 1 illustrates the various readouts possible with epithelial organoid monolayers. Electrodes in the membrane insert to measure TEER. [Figure 3B]The various possible readouts are illustrated using epithelial organoid monolayers. The TEER value increases to values ​​of approximately 100 Ω·cm2 as the monolayers reach confluence. After the monolayers are enriched with intestinal absorptive epithelial cells or a combination of different epithelial cells, the TEER increases to more than 1000 Ω·cm2. Abbreviations: TEER = transepithelial electrical resistance, IEM = intestinal organoid growth medium, eDM = intestinal absorptive epithelial cell differentiation medium, cDM = combined differentiation medium. [Figure 3C] The various possible readouts with epithelial organoid monolayers are illustrated. Monolayers in all media conditions (IEM+4 days IEM / eDM / cDM) are impermeable to Lucifer Yellow. Abbreviations: IEM = Intestinal Organoid Growth Medium, eDM = Intestinal Absorptive Epithelial Cell Differentiation Medium, cDM = Combined Differentiation Medium, Papp = Apparent Permeability Coefficient. [Figure 3D] The various possible readouts with epithelial organoid monolayers are illustrated. Ileal monolayers grown in growth medium express more lysozyme than those grown in either type of differentiation medium (IEM + IEM / eDM / cDM for 4 days). Abbreviations: IEM = Intestinal Organoid Growth Medium, eDM = Intestinal Absorptive Epithelial Cell Differentiation Medium, cDM = Combined Differentiation Medium. [Figure 3E] The various possible readouts with epithelial organoid monolayers are illustrated. Colonic monolayers exhibit different morphologies when exposed to different media conditions (IEM+IEM / eDM / cDM for 4 days) as visualized by H&E, Ki67, Alcian Blue, and MUC2 staining. As expected, monolayers cultured in growth medium are truly proliferative as shown by Ki67 staining. Monolayers differentiated in eDM show columnar epithelium without proliferative cells. Monolayers exposed to cDM are also not proliferative, with more goblet cells appearing. Scale bar = 100 μm. Abbreviations: IEM = intestinal organoid growth medium, eDM = intestinal absorptive epithelial cell differentiation medium, cDM = combined differentiation medium, H&E = hematoxylin and eosin, AB = alcian blue, MUC2 = mucin-2. [Figure 3F]Figure 1 illustrates the various possible readouts using epithelial organoid monolayers. Expression of stem cell marker gene (LGR5), goblet cell marker gene (MUC2), and intestinal absorptive epithelial cell marker gene (ALPI) in colonic monolayers by qRT-PCR. Abbreviations: IEM = intestinal organoid growth medium, eDM = intestinal absorptive epithelial cell differentiation medium, cDM = combined differentiation medium, LGR5 = G protein-coupled receptor with leucine-rich repeats 5, MUC2 = mucin-2, ALPI = intestinal alkaline phosphatase, qRT-PCR = quantitative reverse transcription polymerase chain reaction. [Figure 4-1] Figure 4 illustrates the characterization of normal ileal organoid monolayers cultured in proliferative CNM culture conditions (left), intestinal absorptive epithelial cell eCDM culture conditions (middle) and combined cCDM culture conditions (right) in 96-well Transwell plates. (A) Ileal organoid monolayers stained with hematoxylin and eosin (H&E), Alcian Blue (AB), KI67 and MUC2. Representative images from two independent biological replicates are presented. Scale bars represent 100 μm. [Figure 4-2] (B) Expression of cell-specific genes (i.e., LGR5, MUC2, LYZ, and ALPI1) in ileal organoid monolayers from two independent biological replicates in different culture conditions. For every biological replicate, two technical replicates were measured. Data are presented as mean ± SD for two replicates of two independent experiments. (C) Lysozyme activity in culture supernatants of the apical chamber. (D) Transepithelial electrical resistance (TEER) results of ileal organoid monolayers cultured under proliferation conditions (CNM), intestinal absorptive epithelial cell conditions (eCDM), and combined conditions (cCDM). (E) Lucifer Yellow (LY) permeability results of ileal organoid monolayers cultured under proliferation conditions (CNM), intestinal absorptive epithelial cell conditions (eCDM), and combined conditions (cCDM). LY permeation was measured at the end of the experiment and expressed as apparent permeability coefficient (Papp). C, D and E are presented as the mean ± SD for three technical replicates. [Figure 5-1]Figure 5 illustrates the characterization of normal colon organoid monolayers cultured in proliferative CNM (left), intestinal absorptive epithelial cell eCDM conditions (middle) and combined cCDM (right) in 96-well Transwell plates. (A) Colon organoid monolayers stained with hematoxylin and eosin (H&E), KI67, Alcian blue (AB) and MUC2. Representative images from two independent biological replicates are presented. Scale bars represent 100 μm. [Figure 5-2] (B) Expression of cell-specific genes (i.e., LGR5, MUC2, LYZ, and ALPI) in colonic organoid monolayers from three independent biological replicates in different culture conditions. For every biological replicate, two technical replicates were measured. Data are presented as mean ± SD for two replicates of three independent experiments. (C) Lysozyme activity in culture supernatants of the apical chamber. (D) Transepithelial electrical resistance (TEER) results for colonic organoid monolayers cultured under proliferation conditions (CNM), intestinal absorptive epithelial cell conditions (eCDM), and combined conditions (cCDM). (E) Lucifer Yellow (LY) permeability results for colonic organoid monolayers cultured under proliferation conditions (CNM), intestinal absorptive epithelial cell conditions (eCDM), and combined conditions (cCDM). LY permeation was measured at the end of the experiment and expressed as apparent permeability coefficient (Papp). C, D, and E are presented as mean ± SD for three technical replicates. [Figure 6-1] Figure 6 illustrates the induction of barrier injury in normal colon-derived epithelial monolayers on 96-well Transwell plates by serial titration of inflammatory cytokines in proliferation (CNM) and combination culture conditions (cCDM). All inflammatory cytokines, whether in combination, were used at the final concentrations listed on the graph. (A)-(H) illustrate the transepithelial electrical resistance (TEER) in CNM and cCDM culture conditions. Data are presented as mean ± SD for three technical replicates. [Figure 6-2] See description of Figure 6-1. [Figure 6-3](I) Epithelial monolayer TEER EC50 dose-response curves 24 hours after addition of the combination of inflammatory cytokines in CNM culture conditions. EC50 dose-response curves were calculated by nonlinear regression log(inhibitor) vs. response variable slope (4 parameters). (J) Epithelial monolayer TEER EC50 dose-response curves 24 hours after addition of the combination of inflammatory cytokines in cCDM culture conditions. EC50 dose-response curves were calculated by nonlinear regression log(inhibitor) vs. response variable slope (4 parameters). Note that the 20 ng / ml data point for IFγ / TNF-α treatment was excluded from the EC50 calculation because it did not follow the decreasing trend of TEER in the dose-response curve. [Figure 7A] Figure 1 illustrates the titration of tofacitinib pretreated on normal colon-derived epithelial monolayers on 96-well Transwell plates treated with 1 ng / ml of the inflammatory cytokine combinations IFN-γ / TNF-α / IL-1α (top) and IFN-γ / TNF-α (bottom). Transepithelial electrical resistance (TEER). Data are presented as the mean ± SD of three technical replicates. [Figure 7B] Illustrated is a titration of tofacitinib pretreated on normal colon-derived epithelial monolayers on 96-well Transwell plates treated with 1 ng / ml of the inflammatory cytokine combinations IFN-γ / TNF-α / IL-1α (top) and IFN-γ / TNF-α (bottom). TEER values ​​after 5 and 24 hours compared to the same Transwells before treatment. Data are presented as the mean ± SD of three technical replicates. [Figure 7C] Illustrated is the titration of tofacitinib pretreated on normal colon-derived epithelial monolayers on 96-well Transwell plates treated with 1 ng / ml of the inflammatory cytokine combinations IFN-γ / TNF-α / IL-1α (top) and IFN-γ / TNF-α (bottom). Permeability of the same Transwells 24 hours after treatment. Data are presented as the mean ± SD of three technical replicates. [Figure 7D]Illustrated is a titration of tofacitinib pretreated on normal colon-derived epithelial monolayers on 96-well Transwell plates treated with 1 ng / ml of the inflammatory cytokine combinations IFN-γ / TNF-α / IL-1α (top) and IFN-γ / TNF-α (bottom). Cell viability of the same Transwells 24 hours after treatment. Data are presented as the mean ± SD of three technical replicates. [Figure 7E] Illustrated are titrations of tofacitinib pretreated on normal colon-derived epithelial monolayers on 96-well Transwell plates treated with the inflammatory cytokine combinations IFN-γ / TNF-α / IL-1α and IFN-γ / TNF-α. Dose-response curves for TEER (top), permeability (middle) and cell viability (bottom). EC50 values ​​were calculated by nonlinear regression log(inhibitor) vs response variable slope (4 parameters) of three technical replicates. [Figure 8A] Figure 1 illustrates that pretreatment with tofacitinib inhibits inflammatory cytokine-induced barrier injury in normal colon organoid monolayers on 96-well Transwell plates. Transepithelial electrical resistance (TEER). Data are presented as mean ± SD for three technical replicates. [Figure 8B] Figure 1 illustrates that pretreatment with tofacitinib inhibits inflammatory cytokine-induced barrier injury in normal colon organoid monolayers on 96-well Transwell plates. TEER values ​​after 5 and 24 hours compared to the same Transwells before treatment. Data are presented as mean ± SD for three technical replicates. One-way ANOVA (Dunett's multiple comparison test) was performed on normalized TEER data. *P<0.05, **P<0.01, ***P<0.001. [Figure 8C]Figure 1 illustrates that pretreatment with tofacitinib inhibits inflammatory cytokine-induced barrier injury in normal colon organoid monolayers on 96-well Transwell plates. Permeability of the same Transwells 24 hours after treatment. Data are presented as mean ± SD for three technical replicates. Unpaired t-tests were performed on permeability data. One-way ANOVA (Dunett's multiple comparison test) was performed on permeability data. *P<0.05, **P<0.01, ***P<0.001. [Figure 8D] Figure 1 illustrates that pretreatment with tofacitinib inhibits inflammatory cytokine-induced barrier injury in normal colon organoid monolayers on 96-well Transwell plates. Cell viability of the same Transwell 24 hours after treatment. Data are presented as mean ± SD for three technical replicates. Unpaired t-tests were performed on cell viability data. One-way ANOVA (Dunett's multiple comparison test) was performed on cell viability data. *P<0.05, **P<0.01, ***P<0.001. [Figure 9A] Figure 1 illustrates that pretreatment with tofacitinib inhibits inflammatory cytokine-induced barrier injury in normal ileal organoid monolayers on 96-well Transwell plates. Transepithelial electrical resistance (TEER). Data are presented as mean ± SD for three technical replicates. [Figure 9B] Figure 1 illustrates that pretreatment with tofacitinib inhibits inflammatory cytokine-induced barrier injury in normal ileal organoid monolayers on 96-well Transwell plates. TEER values ​​after 5 and 24 hours compared to the same Transwells before treatment. Data are presented as mean ± SD for three technical replicates. One-way ANOVA (Dunett's multiple comparison test) was performed on normalized TEER data. *P<0.05, **P<0.01, ***P<0.001. [Figure 9C]Figure 1 illustrates that pretreatment with tofacitinib inhibits inflammatory cytokine-induced barrier injury in normal ileal organoid monolayers on 96-well Transwell plates. Permeability of the same Transwells 24 hours after treatment. Data are presented as mean ± SD for three technical replicates. Unpaired t-tests were performed on permeability data. One-way ANOVA (Dunett's multiple comparison test) was performed on permeability data. *P<0.05, **P<0.01, ***P<0.001. [Figure 9D] Figure 1 illustrates that pretreatment with tofacitinib inhibits inflammatory cytokine-induced barrier injury in normal ileal organoid monolayers on 96-well Transwell plates. Cell viability of the same Transwell 24 hours after treatment. Data are presented as mean ± SD for three technical replicates. Unpaired t-tests were performed on cell viability data. One-way ANOVA (Dunett's multiple comparison test) was performed on cell viability data. *P<0.05, **P<0.01, ***P<0.001. [Figure 10A] Figure 1 illustrates the response of organoid epithelial monolayers derived from non-inflamed CD ileum to barrier injury induced by the proinflammatory cytokine combinations IFN-γ / TNF-α / IL-1α (top) and IFN-γ / TNF-α (bottom) with and without pretreatment with tofacitinib. Transepithelial electrical resistance (TEER). Data are presented as mean ± SD for three technical replicates. [Figure 10B]Illustrated are the responses of organoid epithelial monolayers derived from non-inflamed CD ileum to barrier injury induced by the inflammatory cytokine combinations IFN-γ / TNF-α / IL-1α (top) and IFN-γ / TNF-α (bottom) with and without pretreatment with tofacitinib. TEER values ​​after 5 and 24 hours compared to the same Transwells before treatment. Data are presented as mean ± SD for three technical replicates. Two-way ANOVA (Dunett's multiple comparison test) was performed on normalized TEER to compare all conditions to the pretreatment condition. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.1, ns not significant. [Figure 10C] Illustrated are the responses of organoid epithelial monolayers derived from non-inflamed CD ileum to barrier injury induced by the inflammatory cytokine combinations IFN-γ / TNF-α / IL-1α (top) and IFN-γ / TNF-α (bottom) with and without pretreatment with tofacitinib. Permeability of the same Transwell 24 hours after treatment. Data are presented as mean ± SD for three technical replicates. For permeability data, one-way ANOVA (Dunett's multiple comparison test) was used. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.1, ns not significant. [Figure 10D] Illustrated are the responses of organoid epithelial monolayers derived from non-inflamed CD ileum to barrier injury induced by the inflammatory cytokine combinations IFN-γ / TNF-α / IL-1α (top) and IFN-γ / TNF-α (bottom) with and without pretreatment with tofacitinib. Cell viability of the same Transwell 24 hours after treatment. Data are presented as mean ± SD for three technical replicates. For cell viability data, one-way ANOVA (Dunett's multiple comparison test) was used. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.1, ns not significant. [Figure 11A]Figure 1 illustrates the response of organoid epithelial monolayers derived from non-inflamed UC distal colon to barrier injury induced by the proinflammatory cytokine combination IFN-γ / TNF-α / IL-1α with and without pretreatment with tofacitinib. Transepithelial electrical resistance (TEER). Data are presented as mean ± SD for three technical replicates. [Figure 11B] Figure 1 illustrates the response of organoid epithelial monolayers derived from non-inflamed UC distal colon to barrier injury induced by the proinflammatory cytokine combination IFN-γ / TNF-α with and without pretreatment with tofacitinib. Transepithelial electrical resistance (TEER). Data are presented as mean ± SD for three technical replicates. [Figure 11C] Figure 1 illustrates the response of organoid epithelial monolayers derived from non-inflamed UC distal colon to barrier injury induced by the proinflammatory cytokine combination TNF-α / IL-1α with and without pretreatment with tofacitinib. Transepithelial electrical resistance (TEER). Data are presented as mean ± SD for three technical replicates. [Figure 11D] Figure 1 illustrates the response of organoid epithelial monolayers derived from non-inflamed UC distal colon to barrier injury induced by the inflammatory cytokine combination IFN-γ / TNF-α / IL-1α with and without pretreatment with tofacitinib. TEER values ​​after 5 and 24 hours compared to the same Transwells before treatment. Data are presented as mean ± SD for three technical replicates. Two-way ANOVA (Dunett's multiple comparison test) was performed on normalized TEER to compare all conditions to the pretreatment condition. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.1, ns not significant. [Figure 11E]Figure 1 illustrates the response of organoid epithelial monolayers derived from non-inflamed UC distal colon to barrier injury induced by the inflammatory cytokine combination IFN-γ / TNF-α with and without pretreatment with tofacitinib. TEER values ​​after 5 and 24 hours compared to the same Transwells before treatment. Data are presented as mean ± SD for three technical replicates. Two-way ANOVA (Dunett's multiple comparison test) was performed on normalized TEER to compare all conditions to the pretreatment condition. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.1, ns not significant. [Figure 11F] Figure 1 illustrates the response of organoid epithelial monolayers derived from non-inflamed UC distal colon to barrier injury induced by the inflammatory cytokine combination TNF-α / IL-1α with and without pretreatment with tofacitinib. TEER values ​​after 5 and 24 hours compared to the same Transwells before treatment. Data are presented as mean ± SD for three technical replicates. Two-way ANOVA (Dunett's multiple comparison test) was performed on normalized TEER to compare all conditions to the pretreatment condition. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.1, ns not significant. [Figure 11G] Figure 1 illustrates the response of organoid epithelial monolayers derived from non-inflamed UC distal colon to barrier injury induced by the inflammatory cytokine combination IFN-γ / TNF-α / IL-1α with and without pretreatment with tofacitinib. Permeability of the same Transwell 24 hours after treatment. Data are presented as mean ± SD for three technical replicates. For permeability data, one-way ANOVA (Dunett's multiple comparison test) was used. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.1, ns not significant. [Figure 11H]Figure 1 illustrates the response of organoid epithelial monolayers derived from non-inflamed UC distal colon to barrier injury induced by the inflammatory cytokine combination IFN-γ / TNF-α with and without pretreatment with tofacitinib. Permeability of the same Transwell 24 hours after treatment. Data are presented as mean ± SD for three technical replicates. For permeability data, one-way ANOVA (Dunett's multiple comparison test) was used. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.1, ns not significant. [Figure 11I] Figure 1 illustrates the response of organoid epithelial monolayers derived from non-inflamed UC distal colon to barrier injury induced by the inflammatory cytokine combination TNF-α / IL-1α with and without pretreatment with tofacitinib. Permeability of the same Transwell 24 hours after treatment. Data are presented as mean ± SD for three technical replicates. For permeability data, one-way ANOVA (Dunett's multiple comparison test) was used. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.1, ns not significant. [Figure 11J] Figure 1 illustrates the response of organoid epithelial monolayers derived from non-inflamed UC distal colon to barrier injury induced by the inflammatory cytokine combination IFN-γ / TNF-α / IL-1α with and without pretreatment with tofacitinib. Cell viability of the same Transwell 24 hours after treatment. Data are presented as mean ± SD for three technical replicates. One-way ANOVA (Dunett's multiple comparison test) was used for cell viability data. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.1, ns not significant. [Figure 11K]Figure 1 illustrates the response of organoid epithelial monolayers derived from non-inflamed UC distal colon to barrier injury induced by the inflammatory cytokine combination IFN-γ / TNF-α with and without pretreatment with tofacitinib. Cell viability of the same Transwell 24 hours after treatment. Data are presented as mean ± SD for three technical replicates. For cell viability data, one-way ANOVA (Dunett's multiple comparison test) was used. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.1, ns not significant. [Figure 11L] Figure 1 illustrates the response of organoid epithelial monolayers derived from non-inflamed UC distal colon to barrier injury induced by the inflammatory cytokine combination TNF-α / IL-1α with and without pretreatment with tofacitinib. Cell viability of the same Transwell 24 hours after treatment. Data are presented as mean ± SD for three technical replicates. One-way ANOVA (Dunett's multiple comparison test) was used for cell viability data. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.1, ns not significant. [Figure 12A] Illustrated are epithelial monolayers derived from human gastrointestinal organoids: human duodenal epithelial monolayer in CNM (top) and after differentiation with eCDM (bottom). [Figure 12B] Illustrating epithelial monolayers derived from human gastrointestinal organoids. Transepithelial electrical resistance (TEER) of human duodenal epithelial monolayers differentiated in different media at day 9. [Figure 12C] Illustrating an epithelial monolayer derived from human gastrointestinal organoids. Lucifer Yellow (LY) permeability across a human duodenal epithelial monolayer 3 days after differentiation in eCDM. The blank represents LY permeability through a Transwell membrane without an epithelial monolayer. [Figure 12D]Illustrating epithelial monolayers derived from human gastrointestinal organoids. Expression of Pgp1 (ABCB1) and BCRP (ABCG2) genes in human duodenal epithelial monolayers upon proliferation in CNM and differentiation in eCDM. [Figure 12E] Figure 1 illustrates epithelial monolayers derived from human gastrointestinal organoids. Rhodamine 123 (Rho) transport from the basolateral to the apical side of human duodenal epithelial monolayers in growth medium (EM) and differentiation medium (DM) in the presence and absence of a Pgp1 inhibitor (PSC833). [Figure 13] Illustrated are epithelial monolayers derived from human gastrointestinal organoids. Human duodenal and colonic epithelial monolayers in CNM growth conditions (left) and eCDM differentiation conditions (right) were stained with H&E, Ki67, and Alcian blue. [Figure 14] Illustrates the polarization of epithelial monolayers derived from human gastrointestinal organoids. (A) TEER of human gastrointestinal organoid-derived epithelial monolayers differentiated with eCDM. (B) Lucifer Yellow permeability across human gastrointestinal organoid-derived epithelial monolayers differentiated with eCDM. "No monolayer" represents a Transwell membrane without an epithelial monolayer. TEER=transepithelial electrical resistance, eCDM=intestinal absorptive epithelial cell differentiation medium, Papp=apparent permeability coefficient, Gef=gefitinib, A=apical application, B=basolateral application, AB=apical and basolateral application. [Figure 15] Illustrates optimization of culture conditions for growing pulmonary organoid-derived monolayers (Lung-A cultures). (A) Microscopic images illustrating the growth of pulmonary organoid-derived monolayers grown on Transwells coated with ECM (Matrigel) and non-coated Transwells. (B) TEER measurements of pulmonary organoid-derived monolayers grown in culture at different cell seeding densities on Transwells with and without Matrigel coating. [Figure 16A]Illustrating the morphology of lung organoid-derived monolayers grown in different media and formats. Images of H&E stained samples of Lung-A cultures. LuM = Lung Growth Medium, cLuM = Lung Ciliary Differentiation Medium, ALI = Air-Liquid Interface, LLI = Liquid-Liquid Interface. [Figure 16B] Illustrating the morphology of lung organoid-derived monolayers grown in different media and formats. Images of H&E stained samples of Lung-B cultures. LuM = lung growth medium, cLuM = lung ciliary differentiation medium, ALI = air-liquid interface, LLI = liquid-liquid interface. [Figure 16C] Illustrates the morphology of lung organoid-derived monolayers grown in different media and formats. Image of H&E stained sample of Lung-C culture. Ciliated cells are visible on the apical surface of the pseudostratified epithelial layer of cells. LuM=Lung Growth Medium, cLuM=Lung Ciliary Differentiation Medium, ALI=Air-Liquid Interface, LLI=Liquid-Liquid Interface. [Figure 17A] Figure 1 illustrates the characterization of the barrier function of lung organoid-derived monolayers grown in different media and formats by TEER measurements. Illustrative TEER values ​​measured for Lung-A cultures. LuM = Lung Growth Medium, cLuM = Lung Ciliary Differentiation Medium, ALI = Air-Liquid Interface, LLI = Liquid-Liquid Interface. For cultures grown in cLuM medium, differentiation was initiated by changing from LuM medium to cLuM medium at the times indicated for each culture. [Figure 17B] Figure 1 illustrates the characterization of the barrier function of lung organoid-derived monolayers grown in different media and formats by TEER measurements. Illustrative TEER values ​​measured for Lung-B cultures. LuM = Lung Growth Medium, cLuM = Lung Ciliary Differentiation Medium, ALI = Air-Liquid Interface, LLI = Liquid-Liquid Interface. For cultures grown in cLuM medium, differentiation was initiated by changing from LuM medium to cLuM medium at the times indicated for each culture. [Figure 17C]Figure 1 illustrates characterization of the barrier function of lung organoid-derived monolayers grown in different media and formats by TEER measurements. Illustrative TEER values ​​measured for Lung-C cultures. LuM = Lung Growth Medium, cLuM = Lung Ciliary Differentiation Medium, ALI = Air-Liquid Interface, LLI = Liquid-Liquid Interface. For cultures grown in cLuM medium, differentiation was initiated by changing from LuM medium to cLuM medium at the times indicated for each culture. [Figure 18A] Figure 1 illustrates the characterization of the permeability of lung organoid-derived monolayers to Lucifer Yellow. Schematic of the experimental setup for the Lucifer Yellow permeability assay. Lucifer Yellow permeability was measured across lung organoid-derived monolayers grown in various culture conditions. [Figure 18B] Characterization of the permeability of lung organoid-derived monolayers to Lucifer Yellow is illustrated. Lucifer Yellow permeability was measured across lung organoid-derived monolayers grown in various culture conditions. LuM = Lung Growth Medium, cLuM = Lung Ciliary Differentiation Medium, ALI = Air-Liquid Interface, LLI = Liquid-Liquid Interface. Lucifer Yellow permeability was measured 4 or 8 days after initiation of differentiation in cultures grown in cLuM medium and at the corresponding time points in cultures grown in LuM medium. Lucifer Yellow permeability of Lung-A cultures grown in various conditions is illustrated. [Figure 18C] Characterization of the permeability of lung organoid-derived monolayers to Lucifer Yellow is illustrated. Lucifer Yellow permeability was measured across lung organoid-derived monolayers grown in various culture conditions. LuM = Lung Growth Medium, cLuM = Lung Ciliary Differentiation Medium, ALI = Air-Liquid Interface, LLI = Liquid-Liquid Interface. Lucifer Yellow permeability was measured 4 or 8 days after initiation of differentiation in cultures grown in cLuM medium and at the corresponding time points in cultures grown in LuM medium. Lucifer Yellow permeability of Lung-B cultures grown in various conditions is illustrated. [Figure 18D]Characterization of the permeability of lung organoid-derived monolayers to Lucifer Yellow is illustrated. Lucifer Yellow permeability was measured across lung organoid-derived monolayers grown in various culture conditions. LuM = lung growth medium, cLuM = lung ciliary differentiation medium, ALI = air-liquid interface, LLI = liquid-liquid interface. Lucifer Yellow permeability was measured 4 or 8 days after initiation of differentiation in cultures grown in cLuM medium and at the corresponding time points in cultures grown in LuM medium. Lucifer Yellow permeability of lung-C cultures grown in various conditions is illustrated. [Figure 19A] Characterization of lung organoid-derived monolayers grown in different culture conditions is illustrated. LuM = lung growth medium, cLuM = lung ciliary differentiation medium, ALI = air-liquid interface, LLI = liquid-liquid interface. Expression of different genes that are markers of specific cell types is shown: KRT5 (lung basal cell marker). Dx+4 / 8 is the measurement after 4 or 8 days of differentiation for cultures grown in cLuM medium or the corresponding time point for undifferentiated cultures grown in LuM medium only. Gene expression was measured by RT-qPCR for lung-A, lung-B, and lung-C organoid-derived monolayers. The order of the bars follows the order indicated in the figure legend. The order of the bars is: Dx+4 LuM LLI, Dx+8 LuM LLI, Dx+4 LuM ALI, Dx+8 LuM ALI, Dx+4 cLuM LLI, Dx+8 cLuM LLI, Dx+4 cLuM ALI, Dx+8 cLuM ALI. [Figure 19B]Characterization of lung organoid-derived monolayers grown in different culture conditions is illustrated. LuM = lung growth medium, cLuM = lung ciliary differentiation medium, ALI = air-liquid interface, LLI = liquid-liquid interface. Expression of different genes that are markers of specific cell types is shown: SPDEF (goblet cell marker). Dx+4 / 8 is the measurement 4 or 8 days after differentiation for cultures grown in cLuM medium or the corresponding time point for undifferentiated cultures grown in LuM medium only. Gene expression was measured by RT-qPCR for lung-A, lung-B, and lung-C organoid-derived monolayers. The order of the bars follows the order indicated in the figure legend. The order of the bars is: Dx+4 LuM LLI, Dx+8 LuM LLI, Dx+4 LuM ALI, Dx+8 LuM ALI, Dx+4 cLuM LLI, Dx+8 cLuM LLI, Dx+4 cLuM ALI, Dx+8 cLuM ALI. [Figure 19C] Illustrates the characterization of lung organoid-derived monolayers grown in different culture conditions. LuM = lung growth medium, cLuM = lung ciliary differentiation medium. Expression of different genes that are markers of specific cell types is shown: KRT5 (lung basal cell marker). Gene expression was measured by RT-qPCR for lung organoid cultures corresponding to lung-A, lung-B, and lung-C organoid-derived monolayers. The order of the bars follows the order indicated in the figure legend. The order of the bars is as follows: 14d LuM, 21d LuM, 28d LuM, 7d LuM + 7d cLuM, 7d LuM + 14d cLuM, 7d LuM + 21d cLuM. [Figure 19D]Illustrates the characterization of lung organoid-derived monolayers grown in different culture conditions. LuM = lung growth medium, cLuM = lung ciliary differentiation medium. Expression of different genes that are markers of specific cell types is shown: SPDEF (goblet cell marker). Gene expression was measured by RT-qPCR for lung organoid cultures corresponding to lung-A, lung-B, and lung-C organoid-derived monolayers. The order of the bars follows the order indicated in the figure legend. The order of the bars is as follows: 14d LuM, 21d LuM, 28d LuM, 7d LuM + 7d cLuM, 7d LuM + 14d cLuM, 7d LuM + 21d cLuM. [Figure 19E] Characterization of lung organoid-derived monolayers grown in different culture conditions is illustrated. LuM = lung growth medium, cLuM = lung ciliated differentiation medium, ALI = air-liquid interface, LLI = liquid-liquid interface. Expression of different genes that are markers of specific cell types is shown: FOXJ1 (ciliated cell marker). Dx+4 / 8 is measured 4 or 8 days after differentiation for cultures grown in cLuM medium, or the corresponding time point for undifferentiated cultures grown in LuM medium only. Gene expression was measured by RT-qPCR for lung-A, lung-B, and lung-C organoid-derived monolayers. The order of the bars follows the order indicated in the figure legend. The order of the bars is: Dx+4 LuM LLI, Dx+8 LuM LLI, Dx+4 LuM ALI, Dx+8 LuM ALI, Dx+4 cLuM LLI, Dx+8 cLuM LLI, Dx+4 cLuM ALI, Dx+8 cLuM ALI. [Figure 19F]Characterization of lung organoid-derived monolayers grown in different culture conditions is illustrated. LuM = lung growth medium, cLuM = lung ciliary differentiation medium, ALI = air-liquid interface, LLI = liquid-liquid interface. Expression of different genes that are markers of specific cell types is shown: SFTPA1 (alveolar marker). Dx+4 / 8 is the measurement 4 or 8 days after differentiation for cultures grown in cLuM medium or the corresponding time point for undifferentiated cultures grown in LuM medium only. Gene expression was measured by RT-qPCR for lung-A, lung-B, and lung-C organoid-derived monolayers. The order of the bars follows the order indicated in the figure legend. The order of the bars is: Dx+4 LuM LLI, Dx+8 LuM LLI, Dx+4 LuM ALI, Dx+8 LuM ALI, Dx+4 cLuM LLI, Dx+8 cLuM LLI, Dx+4 cLuM ALI, Dx+8 cLuM ALI. [Figure 19G] Illustrates the characterization of lung organoid-derived monolayers grown in different culture conditions. LuM = lung growth medium, cLuM = lung ciliary differentiation medium. Expression of different genes that are markers of specific cell types is shown: FOXJ1 (ciliated cell marker). Gene expression was measured by RT-qPCR for lung organoid cultures corresponding to lung-A, lung-B, and lung-C organoid-derived monolayers. The order of the bars follows the order indicated in the figure legend. The order of the bars is as follows: 14d LuM, 21d LuM, 28d LuM, 7d LuM + 7d cLuM, 7d LuM + 14d cLuM, 7d LuM + 21d cLuM. [Figure 19H]Characterization of lung organoid-derived monolayers grown in various culture conditions is illustrated. Expression of the transport protein OCTN1 was also measured. LuM = lung growth medium, cLuM = lung cilia differentiation medium, ALI = air-liquid interface, LLI = liquid-liquid interface, Dx+4 / 8 is measured 4 or 8 days after differentiation for cultures grown in cLuM medium or the corresponding time point for undifferentiated cultures grown in LuM medium only. Gene expression was measured by RT-qPCR for lung-A, lung-B, and lung-C organoid-derived monolayers. The order of the bars follows the order indicated in the figure legend. The order of the bars is: Dx+4 LuM LLI, Dx+8 LuM LLI, Dx+4 LuM ALI, Dx+8 LuM ALI, Dx+4 cLuM LLI, Dx+8 cLuM LLI, Dx+4 cLuM ALI, Dx+8 cLuM ALI. [Figure 19I] Characterization of lung organoid-derived monolayers grown in various culture conditions is illustrated. Expression of the transport protein MRP1 was also measured. LuM = lung growth medium, cLuM = lung cilia differentiation medium, ALI = air-liquid interface, LLI = liquid-liquid interface, Dx+4 / 8 is measured 4 or 8 days after differentiation for cultures grown in cLuM medium or the corresponding time point for undifferentiated cultures grown in LuM medium only. Gene expression was measured by RT-qPCR for lung-A, lung-B, and lung-C organoid-derived monolayers. The order of the bars follows the order indicated in the figure legend. The order of the bars is: Dx+4 LuM LLI, Dx+8 LuM LLI, Dx+4 LuM ALI, Dx+8 LuM ALI, Dx+4 cLuM LLI, Dx+8 cLuM LLI, Dx+4 cLuM ALI, Dx+8 cLuM ALI. [Figure 19J]Characterization of lung organoid-derived monolayers grown in various culture conditions is illustrated. Expression of the transport protein OCTN1 was also measured. LuM = lung growth medium, cLuM = lung ciliary differentiation medium. Gene expression was measured by RT-qPCR for lung organoid cultures corresponding to lung-A, lung-B, and lung-C organoid-derived monolayers. The order of the bars follows the order indicated in the figure legend. The order of the bars is as follows: 14d LuM, 21d LuM, 28d LuM, 7d LuM + 7d cLuM, 7d LuM + 14d cLuM, 7d LuM + 21d cLuM. [Figure 19K] Characterization of lung organoid-derived monolayers grown in various culture conditions is illustrated. Expression of the transport protein MRP1 was also measured. LuM = lung growth medium, cLuM = lung ciliary differentiation medium. Gene expression was measured by RT-qPCR for lung organoid cultures corresponding to lung-A, lung-B, and lung-C organoid-derived monolayers. The order of the bars follows the order indicated in the figure legend. The order of the bars is as follows: 14d LuM, 21d LuM, 28d LuM, 7d LuM + 7d cLuM, 7d LuM + 14d cLuM, 7d LuM + 21d cLuM. [Figure 20A] 1 illustrates the characterization of transport activity of lung organoid-derived monolayers in an "accumulation" assay format. Lung-C cultures were used for these experiments. The timing of cell seeding, timing of initiation of air-liquid interface culture, and time points at which calcein AM transport assays were performed are illustrated. [Figure 20B] FIG. 1 illustrates the characterization of transport activity of lung organoid-derived monolayers in an "accumulation" assay format. Lung-C cultures were used for these experiments. A schematic diagram of the cell culture format is illustrated. [Figure 20C] 1 illustrates the characterization of transport activity of lung organoid-derived monolayers in an "accumulation" assay format. Lung-C cultures were used for these experiments. A schematic diagram of the "accumulation" assay format is illustrated. [Figure 20D]Characterization of transport activity of lung organoid-derived monolayers in an "accumulation" assay format is illustrated. Lung-C cultures were used for these experiments. Fluorescence measurements of intracellularly accumulated calcein AM are illustrated for Lung-C cultures grown in LuM medium in LLI and ALI culture formats in the presence of MK571 (MRP1 specific inhibitor) and PSC833 (P-gp specific inhibitor). Samples incubated with PBS only (no calcein AM) were used as negative controls. RFU stands for relative fluorescence units. [Figure 21A] 20A and 20B illustrate the characterization of the transport activity of pulmonary organoid-derived monolayers in a "pulse-chase" assay format. These experiments use lung-C cultures, and cells are grown as described in FIG. 20A and FIG. 20B. Schematic diagram of the "pulse-chase" assay format is illustrated. [Figure 21B] 2 illustrates the characterization of the transport activity of lung organoid-derived monolayers in a "pulse-chase" assay format. Lung-C cultures were used for these experiments, and cells were grown as described in FIG. 20A and FIG. 20B. Illustrates the intracellular relative fluorescence of calcein AM measured for lung monolayer cultures grown in LuM medium in LLI format. Samples incubated with PBS (without calcein AM) were included as negative controls along with blank samples. T=0 is the fluorescence measured for cultures at 0 minutes after calcein AM was removed from the apical and basolateral media. T=2 is the fluorescence measured for cultures incubated for 2 hours at 37° C. in fresh medium without calcein AM. [Figure 21C]2 illustrates the characterization of the transport activity of lung organoid-derived monolayers in a "pulse-chase" assay format. Lung-C cultures were used for these experiments, and cells were grown as described in FIG. 20A and FIG. 20B. Illustrates the intracellular relative fluorescence of calcein AM measured for lung monolayer cultures grown in LuM medium in ALI format. Samples incubated with PBS (without calcein AM) were included as negative controls along with blank samples. T=0 is the fluorescence measured for cultures at 0 minutes after calcein AM was removed from the apical and basolateral media. T=2 is the fluorescence measured for cultures incubated for 2 hours at 37° C. in fresh medium without calcein AM. [Figure 21D] 20A and 20B illustrate the characterization of the transport activity of pulmonary organoid-derived monolayers in a "pulse-chase" assay format. These experiments use lung-C cultures, and cells are grown as described in Figure 20A and Figure 20B. 20B illustrates the relative fluorescence of calcein AM measured in the medium of the apical compartment for pulmonary organoid-derived monolayers grown in ALI or LLI format at t=2. [Figure 21E] 20A and 20B illustrate the characterization of the transport activity of pulmonary organoid-derived monolayers in a "pulse-chase" assay format. These experiments use lung-C cultures, and cells are grown as described in Figure 20A and Figure 20B. 20A illustrates the relative fluorescence of calcein AM measured in the medium of the basolateral compartment for pulmonary organoid-derived monolayers grown in ALI or LLI format at t=2. [Figure 22] Optimization of culture conditions for the growth of renal organoid-derived monolayers is illustrated. (A) TEER measurements of renal organoid-derived monolayers grown at different cell seeding densities in culture on Matrigel-coated Transwells. (B) TEER measurements of renal organoid-derived monolayers grown at different cell seeding densities in culture on Matrigel-coated and non-coated Transwells. [Figure 23A]Illustrating the morphology of renal organoid-derived monolayers grown in various culture media. Images of H&E stained samples of Kidney-A cultures. KEM = kidney proliferation medium, KDM = kidney differentiation medium, DAC = KEM supplemented with 1 μM decitabine on day 2. [Figure 23B] Illustrating the morphology of renal organoid-derived monolayers grown in various culture media. Images of H&E stained samples of Kidney-B cultures. KEM = kidney proliferation medium, KDM = kidney differentiation medium, DAC = KEM supplemented with 1 μM decitabine on day 2. [Figure 23C] Illustrating the morphology of renal organoid-derived monolayers grown in various culture media. Images of H&E stained samples of Kidney-C cultures. KEM = kidney proliferation medium, KDM = kidney differentiation medium, DAC = KEM supplemented with 1 μM decitabine on day 2. [Figure 24A] Figure 1 illustrates the characterization of the barrier function of lung organoid-derived monolayers grown in different media by TEER measurements. TEER values ​​measured for kidney-A cultures are illustrated. KEM = kidney proliferation medium, KDM = kidney differentiation medium. [Figure 24B] Figure 1 illustrates the characterization of the barrier function of lung organoid-derived monolayers grown in different media by TEER measurements. TEER values ​​measured for kidney-B cultures are illustrated. KEM = kidney proliferation medium, KDM = kidney differentiation medium. [Figure 24C] Figure 1 illustrates the characterization of the barrier function of lung organoid-derived monolayers grown in different media by TEER measurements. TEER values ​​measured for kidney-C cultures are illustrated. KEM = kidney proliferation medium, KDM = kidney differentiation medium. [Figure 25A] Figure 1 illustrates characterization of the permeability of lung organoid-derived monolayers to Lucifer Yellow. Figure 2 illustrates the permeability of kidney-A cultures grown in various conditions. KEM = kidney growth medium, D4 KDM = KEM changed to kidney differentiation medium (KDM) 4 days after seeding, DAC = KEM supplemented with 1 μM decitabine on day 2. [Figure 25B]Figure 1 illustrates characterization of the permeability of lung organoid-derived monolayers to Lucifer Yellow. Figure 2 illustrates the permeability of kidney-B cultures grown in various conditions. KEM = kidney growth medium, D4 KDM = KEM changed to kidney differentiation medium (KDM) 4 days after seeding, DAC = KEM supplemented with 1 μM decitabine on day 2. [Figure 25C] Characterization of the permeability of lung organoid-derived monolayers to Lucifer Yellow is illustrated. Lucifer Yellow permeability of kidney-C cultures grown in various conditions is illustrated. KEM = kidney growth medium, D4 KDM = KEM changed to kidney differentiation medium (KDM) 4 days after seeding, DAC = KEM supplemented with 1 μM decitabine on day 2. [Figure 26A] Illustrates the characterization of gene expression in renal organoid-derived monolayers grown in different culture conditions. Expression of different genes that are markers of specific cell types is shown: ABCC4 (proximal tubule marker). Gene expression was measured by RT-qPCR for Lung-A, Lung-B, and Lung-C organoid-derived monolayers. The order of the bars follows the order indicated in the figure legend. The order of the bars is as follows: KEM, DAC, KDM. [Figure 26B] Illustrates the characterization of gene expression in renal organoid-derived monolayers grown in different culture conditions. Expression of different genes that are markers of specific cell types is shown: PAX8 (renal epithelial marker). Gene expression was measured by RT-qPCR for Lung-A, Lung-B, and Lung-C organoid-derived monolayers. The order of the bars follows the order indicated in the figure legend. The order of the bars is as follows: KEM, DAC, KDM. [Figure 26C]Illustrates the characterization of gene expression in kidney organoid-derived monolayers grown in different culture conditions. Shown is the expression of different genes that are markers of specific cell types: ABCC4 (proximal tubule marker). 4d / 8d KDM: measurements after 4 or 8 days of culture in kidney differentiation medium (KDM). Gene expression was measured by RT-qPCR in lung organoid cultures corresponding to lung-A, lung-B, and lung-C organoid-derived monolayers. The order of the bars follows the order indicated in the figure legend. The order of the bars is as follows: KEM, 4d KDM, 8d KDM. [Figure 26D] Illustrates the characterization of gene expression in kidney organoid-derived monolayers grown in different culture conditions. Shown is the expression of different genes that are markers of specific cell types: PAX8 (renal epithelial marker). 4d / 8d KDM: measurements after 4 or 8 days of culture in kidney differentiation medium (KDM). Gene expression was measured by RT-qPCR in lung organoid cultures corresponding to lung-A, lung-B, and lung-C organoid-derived monolayers. The order of the bars follows the order indicated in the figure legend. The order of the bars is as follows: KEM, 4d KDM, 8d KDM. [Figure 26E] Illustrates the characterization of gene expression in kidney organoid-derived monolayers grown in different culture conditions. Expression of different genes that are markers of specific cell types is shown: CLDN10 (Henle's loop marker). Gene expression was measured by RT-qPCR for lung-A, lung-B, and lung-C organoid-derived monolayers. The order of the bars follows the order indicated in the figure legend. The order of the bars is as follows: KEM, DAC, KDM. [Figure 26F]Illustrates the characterization of gene expression in kidney organoid-derived monolayers grown in different culture conditions. Shown is the expression of different genes that are markers of specific cell types: CLDN10 (Henle's loop marker). 4d / 8d KDM: measurements after 4 or 8 days of culture in kidney differentiation medium (KDM). Gene expression was measured by RT-qPCR for lung organoid cultures corresponding to lung-A, lung-B, and lung-C organoid-derived monolayers. The order of the bars follows the order indicated in the figure legend. The order of the bars is as follows: KEM, 4d KDM, 8d KDM. [Figure 26G] Illustrates the characterization of gene expression in renal organoid-derived monolayers grown in different culture conditions. Expression of different genes that are markers of specific cell types is shown: AQP3 (collecting duct marker). Gene expression was measured by RT-qPCR for Lung-A, Lung-B, and Lung-C organoid-derived monolayers. The order of the bars follows the order indicated in the figure legend. The order of the bars is as follows: KEM, DAC, KDM. [Fig. 26H] Illustrates the characterization of gene expression in kidney organoid-derived monolayers grown in various culture conditions. Expression of transport proteins OCT2, MATE1, and MATE2-K are also shown. Gene expression was measured by RT-qPCR for lung-A, lung-B, and lung-C organoid-derived monolayers. The order of the bars follows the order indicated in the figure legend. The order of the bars is as follows: KEM, DAC, KDM. [Figure 26I] Illustrates the characterization of gene expression in kidney organoid-derived monolayers grown in various culture conditions. Also shown is the expression of transport proteins OCT2, MATE1, and MATE2-K. 4d / 8d KDM: measurements after 4 or 8 days of culture in kidney differentiation medium (KDM). Gene expression was measured by RT-qPCR in lung organoid cultures corresponding to lung-A, lung-B, and lung-C organoid-derived monolayers. The order of the bars follows the order indicated in the figure legend. The order of the bars is as follows: KEM, 4d KDM, 8d KDM. [Figure 27]Figure 1 illustrates fluorescence measurements of accumulated intracellular calcein AM in the presence or absence of PSC833 (a P-gp specific inhibitor) for kidney-C cultures grown in KEM medium with or without 1 uM decitabine (DAC) added 2 days after seeding. Samples incubated with PBS only (no calcein AM) served as negative controls. RFU stands for relative fluorescence units. [Figure 28] Figure 1 illustrates fluorescence measurements of intracellular rhodamine 123 accumulation in the presence or absence of PSC833 (a P-gp specific inhibitor) and decinium-22 (an OCT2 specific inhibitor) for kidney-C cultures grown in KEM medium with or without 1 uM decitabine (DAC) added 2 days after seeding. Samples incubated with PBS only (no calcein AM) served as negative controls. RFU stands for relative fluorescence units. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0256] The present invention further provides the following numbered embodiments:

[0257] 1. A method for obtaining an organoid-derived monolayer, comprising the steps of: i. digesting or dissociating one or more organoids into a suspension of single cells and / or organoid fragments; ii. seeding a semipermeable membrane with the suspension; and iii. Culturing the cells and / or organoid fragments in the presence of growth medium until a monolayer is formed.

[0258] 2.iv. Culturing the monolayer in the presence of differentiation medium 2. The method of embodiment 1, further comprising:

[0259] 3. The monolayer has a transepithelial electrical resistance (TEER) of about 100 Ω cm 2 3. The method of embodiment 1 or embodiment 2, wherein the cells are cultured in the presence of a growth medium until the cell culture medium reaches a concentration of 0.1 mM.

[0260] 4. The method of embodiment 2 or embodiment 3, wherein the TEER of the monolayer is further increased during the step of culturing the monolayer in the presence of the differentiation medium.

[0261] 5. The TEER of the monolayer is 500 Ω cm during the step of culturing the monolayer in the presence of the differentiation medium. 2 Super, 600Ω cm 2 Super, 700Ω cm 2 Super, 800Ω cm 2 Super, 900Ω cm 2 Super, 1000Ω cm 2 Super, 1100Ω cm 2 Super, 1200Ω cm 2 Super, 1300Ω cm 2 Ultra, 1400Ω cm 2 or greater than 1500 Ω cm 2 5. The method of embodiment 4, wherein the method reaches above

[0262] 6. The method of any one of the preceding embodiments, wherein the growth medium comprises a receptor tyrosine kinase ligand, a BMP inhibitor and a Wnt agonist, and optionally, nicotinamide and a p38 MAPK inhibitor, such as SB202190.

[0263] 7. The method of any one of embodiments 2 to 6, wherein the differentiation medium comprises a Notch inhibitor, an EGFR pathway inhibitor and a Wnt agonist.

[0264] 8. The method according to any one of embodiments 2 to 6, wherein the differentiation medium comprises a Wnt agonist and a Wnt secretion inhibitor.

[0265] 9. The method according to any one of embodiments 6 to 8, wherein the receptor tyrosine kinase ligand is a ligand of RTK class I (EGF receptor family) (ErbB family), a ligand of RTK class II (insulin receptor family), a ligand of RTK class IV (FGF receptor family), or a ligand of RTK class VI (HGF receptor family).

[0266] 10. The method of embodiment 9, wherein the receptor tyrosine kinase ligand is selected from the group consisting of epidermal growth factor (EGF), neuregulin, fibroblast growth factor (FGF), hepatocyte growth factor (HGF), and insulin-like growth factor (IGF).

[0267] 11. The method of any one of embodiments 6 to 10, wherein the BMP inhibitor is selected from the group consisting of noggin, sclerostin, chordin, CTGF, follistatin, gremlin, tsg, sog, LDN193189 or dorsomorphin.

[0268] 12. The method of any one of embodiments 6 to 11, wherein the Wnt agonist is selected from the group consisting of Respondin, Wnt conditioned medium and Wnt substitute.

[0269] 13. The method of any one of embodiments 7 to 12, wherein the Notch inhibitor is a gamma secretase inhibitor, optionally selected from the group consisting of DAPT, dibenzazepine (DBZ), benzodiazepine (BZ) and LY-411575.

[0270] 14. The method of any one of embodiments 7 to 13, wherein the EGFR pathway inhibitor is selected from (1) an EGFR inhibitor such as gefitinib, (2) an EGFR and ErbB2 inhibitor such as afatinib, (3) an inhibitor of the RAS-RAF-MAPK pathway, (4) an inhibitor of the PI3K / AKT pathway, and (5) an inhibitor of the JAK / STAT pathway.

[0271] 15. The method of embodiment 14, wherein the EGFR pathway inhibitor is an inhibitor of the RAS-RAF-MAPK pathway, for example a MEK inhibitor such as PD0325901.

[0272] 16. The method according to any one of embodiments 8 to 15, wherein the Wnt secretion inhibitor is a Porc inhibitor, optionally a Porc inhibitor selected from the group consisting of IWP 2, LGK974 and IWP 1.

[0273] 17.i. the monolayer is cultured in the presence of growth medium for at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, or at least 10 days, preferably the monolayer is cultured in the presence of growth medium for 3-9 days; and / or ii. The monolayer is cultured in the presence of differentiation medium for at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days or more, preferably, the monolayer is cultured in the presence of differentiation medium for 4-8 days; 13. The method of any one of the preceding embodiments.

[0274] 18. The method of any one of the preceding embodiments, wherein the monolayer is cultured in the presence of an extracellular matrix.

[0275] 19. An organoid-derived monolayer obtainable or obtainable by a method according to any one of embodiments 1 to 18.

[0276] 20. Transepithelial electrical resistance (TEER) is 100 Ω·cm 2 Ultra-organoid-derived monolayer.

[0277] 21.500 Ω cm 2 Super, 600Ω cm 2 Super, 700Ω cm 2 Super, 800Ω cm 2 Super, 900Ω cm 2 Super, 1000Ω cm 2 Super, 1100Ω cm 2 Super, 1200Ω cm 2 Super, 1300Ω cm 2 Super, 1400Ω cm 2or greater than 1500 Ω cm 2 21. The organoid-derived monolayer of embodiment 20, having a TEER of greater than 1.

[0278] 22. The method or organoid-derived monolayer of any one of the preceding embodiments, wherein said monolayer is derived from the intestinal tract.

[0279] 23. The monolayer comprises the following cell type: Lgr5 + 23. The method of embodiment 22 or the organoid-derived monolayer, comprising one or more of stem cells, intestinal absorptive epithelial cells, goblet cells, Paneth cells and enteroendocrine cells.

[0280] 24. The method or organoid-derived monolayer of any one of the preceding embodiments, wherein said monolayer is derived from a mammal.

[0281] 25. The method or organoid-derived monolayer according to embodiment 24, wherein said monolayer is derived from a human.

[0282] 26. The method or organoid-derived monolayer according to embodiment 25, wherein the human has a digestive disease or disorder, such as inflammatory bowel disease (e.g., Crohn's disease or ulcerative colitis), celiac disease or leaky gut syndrome.

[0283] 27. Use of an organoid-derived monolayer according to any one of embodiments 19 to 26 in an assay for evaluating epithelial viability, metabolic activity, permeability, barrier function integrity and / or transport protein activity.

[0284] 28. A method for identifying a compound capable of modulating epithelial viability, metabolic activity, permeability, barrier integrity and / or transport protein activity, comprising the steps of: i. contacting an organoid-derived monolayer, such as an organoid-derived monolayer described in any one of embodiments 19 to 26, with one or more candidate molecules; ii. Assessing the viability, metabolic activity, permeability and / or barrier function integrity of the organoid-derived monolayer and / or the activity of transport proteins in the organoid-derived monolayer.

[0285] 29. A method for assessing the effect of a compound on epithelial viability, metabolic activity, permeability, barrier integrity and / or transport protein activity, comprising the steps of: i. contacting an organoid-derived monolayer, such as an organoid-derived monolayer described in any one of embodiments 19 to 26, with the compound; and ii. Assessing the viability, metabolic activity, permeability and / or barrier function integrity of the organoid-derived monolayer and / or the activity of transport proteins in the organoid-derived monolayer.

[0286] 30. The method of embodiment 28 or embodiment 29, further comprising contacting the organoid-derived monolayer with one or more inflammatory cytokines.

[0287] 31. The method of embodiment 30, wherein the one or more inflammatory cytokines are selected from the group consisting of IFN-γ, TNF-α, and IL-1α.

[0288] 32. A method for identifying mutations associated with epithelial viability, metabolic activity, permeability, barrier integrity and / or transport protein activity, comprising the steps of: i. assessing the viability, metabolic activity, permeability and / or barrier function integrity of an organoid-derived monolayer, e.g., an organoid monolayer according to any one of embodiments 19 to 26, and / or the activity of transport proteins in the organoid-derived monolayer; ii. Determining the presence of one or more mutations in the genome of one or more cells in the organoid-derived monolayer.

[0289] 33.i. Obtaining an organoid-derived monolayer from the human subject as described in any one of embodiments 1 to 18; and ii. testing the viability, metabolic activity, permeability and / or barrier function integrity of said organoid-derived monolayer and / or the activity of transport proteins in said organoid-derived monolayer. 1. A method for diagnosing a disease or affliction affecting epithelial viability, metabolic activity, permeability, barrier function integrity and / or transport protein activity in a human subject, or determining an increased risk of said disease or affliction, comprising:

[0290] 34. The method of embodiment 33, wherein the reference value is a value obtained from a control, e.g., an organoid-derived monolayer obtained from a healthy human subject.

[0291] 35. The method of embodiment 33 or embodiment 34, wherein the disease or affliction is a disease or disorder of the digestive system, such as inflammatory bowel disease (e.g., Crohn's disease or ulcerative colitis), celiac disease or leaky gut syndrome.

[0292] 36. A method for predicting the likelihood of a patient responding to a candidate compound, comprising the steps of: i. Obtaining an organoid-derived monolayer from the patient, as described in any one of embodiments 1-18; ii. contacting said organoid-derived monolayer with said compound; and iii. Assessing the viability, metabolic activity, permeability and / or barrier function integrity of the organoid-derived monolayer and / or the activity of transport proteins in the organoid-derived monolayer.

[0293] 37. The use or method of any one of embodiments 27 to 36, wherein the step of assessing the integrity of the barrier function of the organoid-derived monolayer comprises measuring the TEER of the organoid-derived monolayer.

[0294] 38. The use or method of any one of embodiments 27 to 37, wherein the step of assessing the permeability of the organoid-derived monolayer comprises measuring the passive diffusivity of a reporter compound across the monolayer.

[0295] 39. The use or method of embodiment 38, wherein said reporter compound is a dye, optionally a fluorescent dye such as Lucifer Yellow.

[0296] 40. The use or method of any one of embodiments 27 to 39, wherein the step of assessing the activity of the transport protein comprises measuring the rate of transport of a substrate of the transport protein across the monolayer membrane, optionally in the presence of an inhibitor of the transport protein.

[0297] 41. The use or method of embodiment 40, wherein the substrate is a dye, such as Rhodamine 123. EXAMPLES

[0298] Example 1. Preparation of epithelial monolayers derived from human normal intestinal organoids. In this protocol, epithelial monolayers are prepared from human normal intestinal organoids, but the protocol can be applied and optimized for other organoid models. Epithelial organoid monolayers are cultured in intestinal organoid growth medium containing Wnt to support stem cell proliferation and represent the cellular composition of intestinal crypts. By modulating the Wnt, Notch, and epidermal growth factor (EGF) pathways, intestinal organoids can be enriched to have various intestinal epithelial fates, including intestinal absorptive epithelial cells, Paneth cells, goblet cells, and enteroendocrine cells. Here, as previously described, after establishing a monolayer in a growth medium, differentiation of intestinal epithelial cells in the monolayer is induced to become more diverse (van Es, JHet al.Wnt signalling induces maturation of Paneth cells in intestinal crypts.Nature Cell Biology.7(4),381-386(2005); van Es, JHet al.Dll1 marks early secretory progenitors in gut crypts that can revert to stem cells upon tissue damage.Nature Cell Biology.14(10),1099-1104(2012); de Lau, WBM, Snel, B., Clevers, H.The R-spondin protein family.Genome Biology.13(3),1-10(2012); Basak, O., Beumer, J., Wiebrands, K., Seno, H., van Oudenaarden, A., Clevers, H.Induced quiescence of Lgr5+ stem cells in intestinal organoids enables differentiation of hormone-producing enteroendocrine cells.Cell Stem Cell.20(2),177-190.e4(2017), Beumer, J. et al.Enteroendocrine cells switch hormone expression along the crypt-to-villus BMP signalling gradient.Nature Cell Biology.20(8),909-916(2018), Yin,X.,Farin,HF,van Es,JH,Clevers,H.,Langer,R.,Karp,JMNiche-independent high-purity cultures of Lgr5+ intestinal stem cells and their progeny.Nature Methods.11(1),106-112(2014)). For screening purposes, depending on the mode of action of the compound of interest, the target cells of the compound of interest, and the experimental conditions, the monolayers can be oriented to the cell composition of choice to measure the effect of the compound on the functional readout. .

[0299] 1. Preparation of Culture Reagents NOTE: All steps are carried out in a biosafety cabinet and following standard guidelines for cell culture. Use UV light for 10 min before starting the biosafety cabinet. Clean the surfaces of the biosafety cabinet with tissue paper soaked in 70% ethanol before and after use. To facilitate the formation of 3D droplets of extracellular matrix (ECM), keep a stock of 96-well, 24-well, and 6-well plates pre-warmed in a 37 °C incubator. 1. Preparation of Basal Medium 1. Prepare Basal Medium (BM) by adding 5 mL of 200 mM glutamine, 5 mL of 1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 5 mL of penicillin / streptomycin (pen / strep) solution (10,000 U / mL or 10,000 μg / mL) to a 500 mL medium bottle of Advanced Dulbecco's Modified Eagle Medium (Ad-DF) with Ham's Nutrient Mixture F-12. Store the basal medium at 4° C. in a refrigerator for at least 4 weeks. 2.Wnt source 1. Prepare Wnt3a conditioned medium (Wnt3aCM) as previously described (Boj, S. F. et al. Forskolin-induced swelling in intestinal organoids: An in vitro assay for assessing drug response in cystic fibrosis patients. Journal of Visualized Experiments. 2017(120), 1-12(2017)). Note: In recent years, next-generation Wnt surrogates (NGS-Wnt) have been generated that also support the growth of human intestinal organoids (Miao, Y. et al. Next-generation surrogate Wnts support organoid growth and deconvolute Frizzled pleiotropy in vivo. Cell Stem Cell. 27(5), 840-851(2020)). 3. Preparation of Intestinal Organoid Basal Medium NOTE: When possible, use growth factors and reagents according to the manufacturer's recommendations. Use small aliquots and avoid freeze-thaw cycles when possible. Functional growth factors are favorable for successful culture of organoids. 1. Prepare enriched 2x Intestinal Organoid Basal Medium (2x IBM) by supplementing BM with 1 µM A83-01, 2.5 mM N-acetylcysteine, 2x B27 supplement, 100 ng / mL human epidermal growth factor (hEGF), 10 nM gastrin, 200 ng / mL hNoggin, and 100 µg / mL antimicrobial preparation for primary cells. 2. Aliquot 2x IBM and freeze at -20° C. for up to 4 months. When needed, thaw aliquots at 4° C. overnight or at room temperature (RT) for several hours. 3. To prepare Intestinal Organoid Growth Medium (IEM, also referred to herein as CNM), supplement 2x IBM with either 50% Wnt3aCM or 50% BM and 0.5 nM NGS-Wnt, 250 ng / mL human Rspondin-3 (hRspo3), 10 mM nicotinamide, and 10 μM SB202190. 4. Preparation of Intestinal Organoid Differentiation Medium Prepare intestinal epithelial cell differentiation medium (eDM) by supplementing 1.2×IBM with 50% BM, 250 ng / mL hRspo3, and 1.5 μM Wnt pathway inhibitor (IWP-2). Store eDM at 4° C. for up to 10 days. Prepare combined differentiation medium (cDM) by supplementing 2.2x IBM with either 40% BM and 10% Wnt3aCM or 50% BM and 0.1 nM NGS-Wnt, 250 ng / mL hRspo3, 10 μM DAPT, and 100 nM PD0325901. Store cDM at 4° C. for up to 10 days. 5. Manipulation of Extracellular Matrix (ECM) NOTE: Prepare the extracellular matrix (ECM) according to the manufacturer's recommendations. 6. Thaw the ECM overnight on ice. Transfer the ECM in the bottle to a 15 mL conical tube pre-chilled at -20°C using a 5 mL pipette pre-chilled at -20°C. Refreeze an aliquot at -20°C once only. Once thawed, store the ECM in the refrigerator at 4°C for up to 7 days. Incubate on ice for at least 30 minutes before use. 7. NOTE: It is advantageous to ensure that the ECM is properly mixed and chilled prior to embedding the crypts or organoids.

[0300] 2. Organoid Culture 1. Subculture of Intestinal Organoids to Prepare Epithelial Monolayers 1. Subculture organoids 3 days prior to harvesting to prepare monolayers. When harvesting organoids for monolayer preparation, resuspend organoids in 1-1.5x starting volume of IEM / ECM to achieve higher density and proliferation potential.

[0301] 3. Preparation of Epithelial Monolayers 1. Epithelial monolayers are cultured on membrane inserts in both 24-well and 96-well plates using the various plate types available. For both sizes, High Throughput System (HTS) membrane inserts are used, which contain a tray with an integrated membrane insert and receiver plate. For the 24-well format, plates with separate removable membrane inserts are used. NOTE: Various membrane types (polyethylene terephthalate (PET) or polycarbonate) and pore sizes (0.4–8.0 µm) are available and can be used depending on experimental needs. Monolayers can be imaged in bright field only with PET membrane inserts. Light-resistant membranes prevent leakage of fluorescence from the apical to the basolateral compartment and may be considered when studying dynamic transport or permeability of fluorescently labeled substrates. The current protocol uses 24-well membrane inserts, but can be adapted to 96-well membrane inserts. Depending on the density, morphology, and size of the organoids, 6 wells of a 6-well plate are sufficient to seed all the membrane inserts in a 24-well plate. 2. Coating Membrane Inserts with ECM NOTE: If there is any doubt about the number of cells, count the cells before coating the inserts to prevent unnecessary coating and loss of expensive membrane inserts. 1. In a biosafety cabinet, place the membrane insert into the support plate. 2+ and Mg 2+ Dilute the ECM 40x with ice-cold Dulbecco's Phosphate Buffered Saline (DPBS) containing 0.1% EDTA and pipette 150 μL of diluted ECM into the apical compartment of each insert. Incubate plates at 37° C. for at least 1 hour. 3. Preparation of Cells for Seeding 1. Pre-warm an aliquot of cell dissociation reagent in a water bath (37° C.). Prepare 2 mL of reagent for each well of a 6-well plate. 2. Transfer the culture plate containing the organoids from the incubator to the biosafety cabinet. Process and passage the organoids as described above. Do not pool multiple tubes into one tube. 3. Add DPBS (Ca) to the tube containing up to 3 organoids from a 6-well plate. 2+ and Mg 2+ Add 10 mL of PBS (not included) to reach 12 mL, pipette up and down 10x with a 10 mL pipette, centrifuge at 85xg for 5 min at 8°C, and aspirate the supernatant without disturbing the organoid pellet. 4. Add 2 mL of pre-warmed cell dissociation reagent per well of the 6-well plate used as starting material and resuspend. Incubate the tube at an angle or horizontally in a 37°C water bath for 5 minutes to prevent organoids from sinking to the bottom of the tube. 5. Pipette up and down 10x using a sterile 5 mL plastic pipette or a P1000 pipette, depending on the total volume of cell dissociation reagent. Observe the organoid suspension under a microscope to see if a mixture of single cells and some cell clumps consisting of 2-4 cells have formed (Figure 3B). If necessary, continue the digestion by repeating steps 3.3.4-3.3.5 until single cells and small cell clumps are visualized in the mixture. NOTE: If possible, avoid completely digesting the organoids down to single cells. It is advantageous to have several small cell groups (e.g., groups of 2–4 cells). 6. Stop cell dissociation by adding BM to the cell suspension until it reaches 12 mL. Centrifuge at 450 x g for 5 min at 8 °C and aspirate the supernatant without disturbing the cell pellet. If working with the same organoid culture in several 15 mL conical tubes, pool the cell pellets and resuspend them in 12 mL of BM. 7. Filter the cell suspension through a 40 μm strainer pre-wetted with BM and collect the flow-through in a 50 mL conical tube. Wash the strainer with 10 mL of BM and collect the flow-through in the same 50 mL conical tube. 8. Transfer the strained cell suspension into two new 15 mL conical tubes. Centrifuge at 450 x g for 5 minutes at 8 °C and aspirate the supernatant without disturbing the cell pellet. Resuspend the cells in 4 mL of IEM (supplemented with 10 μM ROCK inhibitor) per complete culture plate used as starting material. 9. For counting, mix a small amount of cell suspension with trypan blue in a 1:1 ratio. Count live cells (not blue) and calculate the total number of live cells. For small cell clumps, count each individual cell. 10. 3 × 10 per mL of IEM supplemented with 10 μM ROCK inhibitor 6 Prepare a cell suspension containing viable cells. 4. Seeding of Cells onto Polyester Membrane Inserts 1. While holding the plate horizontally, carefully aspirate the DPBS from the ECM-coated inserts (step 3.2.1). Pipette 800 μL of IEM supplemented with ROCK inhibitor into each basolateral compartment. Pipette 150 μL of the cell suspension prepared in step 3.3.10 onto the ECM-coated membrane of the apical compartment. Ensure that at least one well per plate is a "blank" well containing BM only. 2. Once the cells have been deposited onto the membrane, measure the transepithelial electrical resistance (TEER) and image the membrane insert using a microscope as described herein. Place the plate in an incubator at 37°C and 5% CO2. Measure the TEER daily and take images periodically to monitor the formation of a monolayer (Figure 1A-D). 5. Refreshing the monolayer NOTE: To maintain hydrostatic pressure on top of the cells and prevent the cells from being pushed out of the membrane, it is advantageous to refresh the medium every 2-3 days by strictly adhering to the following order: When refreshing the medium, be careful not to damage the monolayer membrane, which is visible when aspirating the medium, with the pipette tip. 1. Remove the medium from the basolateral compartment of the plate containing the membrane inserts. Then carefully aspirate the medium from the apical compartment of the membrane inserts. 2. Drop 150 μL of fresh IEM into each apical compartment, then add 800 μL of fresh IEM to each basolateral compartment. 6. Enrichment of Monolayers Towards Desired Intestinal Epithelial Cell Types 1. Grow the monolayer to confluence in an IEM and place it under 100 Ω cm pressure. 2 Correspond to the surrounding TEER value. Check microscopy to determine if the monolayer is fully formed (Figure 1D) and if there are any holes (as seen in Figures 1B, 1C). 2. Carefully remove the IEM from the basal and apical compartments of the membrane insert and replace it with either eDM or cDM prepared in section 1.4. Culture the monolayer in specific differentiation medium for an additional 3-4 days to obtain organoid cells enriched for the specific cell type desired. Refresh the medium every 2-3 days as described in section 3.4. 3. If necessary, measure TEER daily and acquire images periodically (Figure 2A-Figure 2C). NOTE: The TEER value that indicates that the monolayer is fully organized and concentrated will vary for each organoid culture. Typically, TEER values ​​are above 600 Ω cm after 3 days in differentiation medium. 2 up to 1000 Ω cm 2 and remains stable for 3 to 5 days.

[0302] 4. Representative results If you are passage the organoids to prepare a monolayer, make sure to seed the organoids at a high density and grow them for 3 days to ensure that the organoids are under optimal growth conditions to ensure sufficient cell numbers for seeding the monolayer. Organoids can be harvested for monolayer preparation at the appropriate size and density, typically 6 wells of a 6-well plate (each well containing 200 μL of organoid domes) are sufficient to seed all of the membrane inserts of a 24-well plate. After preparing a single cell suspension with cell dissociation reagent, single cells and small cell clumps should be visible and live cells can be counted. Dead cells stained with trypan blue should be excluded from the count. The single cells and small cell clumps are then seeded onto the membrane inserts as seen in Figure 1A. Monolayer formation can be seen after 1-3 days (Figure 1B, Figure 1C), and the monolayer generally becomes confluent after 3-6 days depending on the organoid culture (Figure 1D). The monolayers remain in growth medium until they become confluent, after which various enrichment media can be used to enrich the monolayers for intestinal absorptive epithelial cells or goblet cells, among others. Figure 2A shows a monolayer cultured for 8 days in growth medium (IEM). When enriched for intestinal absorptive epithelial cells (eDM), a structure similar to that of Figure 2B is seen, whereas monolayers exposed to combination medium (cDM) show a smoother structure (Figure 2C).

[0303] Monolayer formation can be quantitatively followed by measuring the TEER (Figure 3A). A fully confluent monolayer has a TEER value of approximately 100 Ω cm 2 and approximately 1000 Ω cm when exposed to either differentiation medium. 2In all media conditions, the monolayers are impermeable to Lucifer Yellow (0.45 kDa), but when the monolayers are intentionally injured, an increase in the apparent permeability coefficient (Papp) can be seen (Figure 3C). Lysozyme secretion by ileal monolayers cultured in IEM was greater than that by monolayers cultured in IEM until confluent and then cultured in eDM or cDM for an additional 4 days (denoted as + followed by eDM or + followed by cDM) (Figure 3D). Monolayers cultured in IEM, IEM + followed by eDM, or IEM + followed by cDM displayed different morphologies, as observed by H&E staining (Figure 3E). Colon organoid-derived epithelial monolayers in IEM and cDM media have a smooth apical surface, whereas monolayers differentiated into intestinal absorptive epithelial cells in the absence of Wnt display an apical invaginated morphology. Ki67-positive proliferative cells could only be detected under proliferative conditions. When Wnt, Notch, and EGF signaling were inhibited, respectively, Alcian blue staining and staining for MUC2, a mucus produced by goblet cells, were visualized in monolayers differentiated in eDM and more prominently in cDM (Figure 3E). While proliferative cells decreased upon differentiation, the expression levels of goblet cell and intestinal absorptive epithelial cell marker genes increased compared to the expression levels observed in IEM conditions. This was shown by quantification of LGR5, MUC2, and ALPI gene expression, respectively, by qRT-PCR (Figure 3F).

[0304] The protocol described above has also been shown to be effective in generating monolayers from dog and rat intestinal organoids. The TEER of rat organoid-derived monolayers is approximately 20 Ω cm. 2 whereas the TEER of the canine organoid-derived monolayer was 1000 Ω cm 2 reached super.

[0305] Example 2. Establishment, differentiation and characterization of human gastrointestinal epithelial monolayers Currently, the effects of compounds on intestinal permeability and barrier function are studied either with transformed cell lines, such as colon adenocarcinoma cell lines Caco-2, T84 or HT-29, or with primary gastrointestinal epithelial tissue mounted in Ussing chambers. Although cell lines can form differentiated and polarized monolayers containing intestinal absorptive epithelial-like and goblet-like cells, many different enzymes and transporters are abnormally expressed in these cell lines, thus reducing the complexity and physiological relevance. In addition, cell lines are from a single donor and therefore do not represent the heterogeneity of the patient population. If epithelial monolayers were to be prepared from intestinal organoids, they would combine the proliferation of cell lines with the physiological relevance and high patient relevance of primary tissues. Therefore, we attempted to establish monolayers using human ileal and colonic organoids. For this purpose, organoids were digested into single cells and seeded on Transwell membranes in CNM, eCDM and cCDM culture conditions.

[0306] In CNM conditions, H&E staining of epithelial monolayer cross sections showed a simple squamous epithelium in both ileum (Figure 4A) and colon models (Figure 5A). Further histological staining showed the presence of proliferative cells (KI67) and the absence of goblet cells (Alcian blue and MUC2) (Figures 4A and 5A, CNM conditions). Analysis of gene expression of LGR5 and MUC2 genes by RT-qPCR confirmed the histological findings, and the lack of ALPI1 expression indicated the absence of intestinal absorptive epithelial cells in the monolayers generated in CNM conditions (Figures 4B and 5B). Expression of lysozyme (LYZ) was detected in both ileum and colon organoid-derived monolayers (Figures 4B and 5B), and lysozyme activity was measured in the supernatants collected from the apical chamber of the Transwell (Figures 4C and 5C).

[0307] In the ileal and colonic monolayers cultured under eCDM conditions, their morphology changed to simple columnar epithelium, and proliferative cells (KI67 + ) and LGR5 +eCDM culture conditions showed fewer stem cells (Figure 4A, 4B, 5A, and 5B). Alcian blue and MUC2 staining revealed a total absence or limited number of goblet cells in ileal and colonic epithelial monolayers, respectively. This was confirmed by RT-qPCR, which also showed lower MUC2 expression levels in eCDM culture conditions compared to cCDM culture conditions (Figure 4A, 4B, 5A, and 5B). In contrast, ALPI1 expression analysis suggested that intestinal absorptive epithelial cells were strongly enriched in the ileal-derived monolayers in eCDM culture conditions (Figure 4B). Finally, LYZ mRNA levels and lysozyme activity were reduced in eCDM culture conditions compared to CNM culture conditions. This is expected to be due to the inhibition of the WNT pathway in eCDM culture conditions (Figure 4B, 4C, 5B, and 5C).

[0308] In cCDM culture conditions, similar to eCDM, no proliferative or stem cells were observed and LYZ1 expression was reduced (Figure 4B, Figure 4C, Figure 5B, and Figure 5C, cCDM conditions). Furthermore, the expression levels of ALPI1 mRNA indicated reduced differentiation of intestinal absorptive epithelial cells compared to eCDM. However, goblet cells appeared in higher numbers in both ileum- and colon-derived epithelial monolayers, as revealed by both Alcian blue and MUC2 staining and RT-qPCR expression analysis (Figure 4B, Figure 4C, Figure 5B, and Figure 5C).

[0309] The formation and integrity of the epithelial monolayer was assessed by transepithelial electrical resistance (TEER); under CNM culture conditions, TEER was 100–200 Ω cm on days 3–7. 2 The TEER is at least 100 Ω cm 2 After reaching a TEER of 1000 Ω cm, the monolayers were allowed to differentiate and the differentiation was followed up by TEER for another 4 days. Among the culture conditions tested, the TEER remained stable in CNM, whereas the TEER in eCDM and cCDM was approximately 1000 Ω cm. 2The expression of tight junction proteins was increased to 0.05 to 0.1, indicating increased barrier integrity, possibly due to increased expression of tight junction proteins (Figure 4 and Figure 4D). Indeed, RT-qPCR analysis showed that the expression of tight junction protein complexes, such as ZO-1 and OCLN, was altered in ileum-derived monolayers in a 24-well format.

[0310] Next to the TEER measurements (Figures 4D and 5D), the paracellular permeability of the monolayers after 4 days of culture in differentiation medium was assessed by passive diffusion of Lucifer Yellow (LY) from the apical to the basal side. When the monolayers were injured by scratching, LY diffused to the basal side. However, the levels of LY were not comparable to those in blank wells (no monolayer), suggesting that LY was anchored to the monolayer. In both ileum and colon-derived monolayers, no diffusion of LY from the apical to the basal compartment was observed, suggesting that both differentiated and undifferentiated epithelial monolayers were impermeable (Figures 4E and 5E).

[0311] Epithelial monolayer formation and differentiation experiments were performed in at least two biological replicates to assess the reproducibility of the assay. Representative tissue sections stained with KI67, AB (Alcian Blue) and MUC2 are shown in Figures 4A and 5A. Gene expression was analyzed by RT-qPCR and the results are presented in Figures 4B and 5B as the average of at least two biological replicates. Individual measurements of TEER, permeability and lysozyme activity are shown in Figures 4C, 4D, 5C and 5D. As the TEER measurements of the first colon biological replicate in CNM conditions (Figure 5D, middle panel) were not reproduced in the second biological replicate, we performed a third biological replicate. The results were comparable to the first replicate. Spontaneous differentiation in CNM culture conditions or depletion of growth factors such as Wnt in the second replicate may explain the results observed in the second replicate.

[0312] Apart from this latter observation, the results from the biological replicates were comparable, indicating that organoids can be used to establish human epithelial monolayers derived from different GI regions. These epithelial monolayers were polarized and were able to differentiate into intestinal absorptive epithelial cells and mucus-producing goblet cells. Moreover, the barrier integrity of the epithelial monolayers was increased and remained impermeable to LY.

[0313] Example 3. Development of an in vitro biological system to mimic components of IBD pathophysiology and provide a robust readout of barrier function pathways Herein, a screening platform based on organoid-derived epithelial monolayers was developed, optimized and validated to serve as a robust and functional readout of barrier function.

[0314] Organoid-derived epithelial monolayers Despite comparable TEER values ​​between eCDM and cCDM conditions (Figures 4D and 5D), intestinal organoids cultured in cCDM produced more mucus, and therefore CNM and cCDM culture conditions were selected for further assay development of barrier integrity (Figures 4A, 4B, 5A, and 5B).

[0315] To investigate the impact of several inflammatory cytokines on the barrier function of monolayers generated from colon-derived organoids, we titrated the most relevant inflammatory cytokines implicated in IBD (IFN-γ, TNF-α, and IL-1α) and determined the EC values ​​of these cytokines in a 24-h assay window. 50TEER values ​​were obtained (Figure 6A-6H). Because IFN-γ has a profound effect on the induction of barrier damage via the JAK-STAT signaling pathway, we titrated various combinations of the three inflammatory cytokines required to eliminate the synergistic effect of TNF-α and IL-1α in combination with IFN-γ. Titrating these three cytokines from 0.25 ng / ml up to 100 ng / ml resulted in a dose-dependent decrease in TEER in both culture conditions after 5 hours. This effect remained relatively stable up to 24 hours in CNM (Figure 6A), whereas a further decrease in TEER was observed in cCDM, especially at concentrations higher than 2 ng / ml. This suggested a complete loss of barrier integrity, likely due to cell death (Figure 6B). The poor sensitivity of organoid-derived epithelial monolayers cultured in CNM correlated with lower expression of IFN-γ receptor (IFNGR1) compared to cCDM culture conditions.

[0316] The presence of two additional cytokines in combination with IFN-γ resulted in a triple combination of IFN-γ, TNF-α, and IL-1α (EC 50 1.77) and the combination of IFN-γ / TNF-α (EC 50 1.67) and IFN-γ treatment alone (EC 50 The epithelial monolayers became more vulnerable to inflammatory cytokine injury as evidenced by a comparison of the TNF-α / IL-1α / IFN-γ combination (EC 50 1.74) after 5 h, but not after 5 h (Figures 6A-H and 6J, Table 5). cCDM culture conditions were selected for further assay development because they expressed a more physiologically relevant cellular heterogeneity, the combination of three inflammatory cytokines showed a strong IFN-γ-specific effect on barrier integrity, and the dynamic range was increased, thereby expanding the signal window for screening purposes. EC of the combination of three inflammatory cytokines on colonic organoid-derived epithelial monolayers cultured in cCDM50 was determined as 2 ng / ml (FIGS. 6I and 6J) and concentrations around this point were used in follow-up experiments.

[0317] (Table 5)EC 50 EC calculated from dose-response curve 50 TIFF2024525079000011.tif38152

[0318] Tofacitinib protects epithelial monolayers from inflammatory cytokine-induced barrier injury For screening purposes, we evaluated the inhibition of inflammatory cytokine-induced barrier dysfunction by tofacitinib in organoid-derived epithelial monolayers on 96-well Transwell plates. Single organoid cell suspensions derived from colonic organoids were cultured in vitro until epithelial monolayers formed and the TEER was 100 Ω cm. 2 Transwells were seeded in CNM conditions for 3–6 days until the epithelial monolayer barrier integrity was further enhanced (TEER > 1000 Ω cm). 2The culture medium was changed to cCDM until 4 h of incubation. Monolayers were then pretreated with various concentrations of tofacitinib for 1 h, followed by treatment with the inflammatory cytokine cocktail IFN-γ / TNF-α / IL-1α, or IFN-γ / TNF-α at individual final concentrations of 1 ng / ml and 2 ng / ml (Figure 7A). The effect of tofacitinib on barrier integrity was determined by measuring TEER after 5 and 24 h. These measurements were normalized to the TEER value of the same Transwell before the measurement to correct for well-to-well TEER variability (Figure 7B). Paracellular permeability was measured after 24 h by Lucifer Yellow (LY) permeability assay (Figure 7C). Monolayer cell viability was then measured by ATP luminescence assay (CellTiter-Glo 3D) to monitor loss of barrier function due to cell death as opposed to increased paracellular permeability. The results indicated that 2 ng / ml of the inflammatory cytokine cocktail caused complete cell death after 24 h, and the viability of epithelial monolayers treated with 1 ng / ml of the triple- and dual-inflammatory cytokine cocktail combinations was reduced to 20% and 50%, respectively (Figure 7D).

[0319] Treatment of colonic epithelial monolayers with combinations of inflammatory cytokines (IFN-γ / TNF-α / IL-1α or IFN-γ / TNF-α, final concentrations of 1 ng / ml and 2 ng / ml, respectively) resulted in a decrease and complete loss of barrier integrity after 5 and 24 hours, respectively. Pretreatment of epithelial monolayers with increasing concentrations of tofacitinib maintained the integrity of barrier function for both cytokine combinations at concentrations above 3 μM (Figures 7A and 7B). Consistent with TEER, the apparent permeability coefficient of LY also decreased with increasing concentrations of tofacitinib above 3 μM (Figure 7C). Cell viability measurements indicated that a final concentration of 2 ng / ml of the inflammatory cytokine cocktail was lethal to epithelial monolayers, whereas 1 ng / ml was well tolerated. However, pretreatment with tofacitinib at concentrations above 3 μM prevented cytokine-induced cell death (Figure 7D). The above experimental conditions were reproduced by repeating the same experiment with an epithelial monolayer derived from the same normal colon and, as in the previous experiment, expanded to an organoid epithelial monolayer derived from the normal ileum of the same donor.

[0320] Epithelial monolayers were incubated with high concentration (10 μM), EC 50 After pretreatment with peripheral (2 μM) and low (0.1 μM) concentrations of tofacitinib (FIG. 7E and Table 6), barrier injury was induced 1 h later with 1 ng / ml of each of a combination of inflammatory cytokines (FIGS. 8 and 9). To highlight the specificity of IFN-γ in inducing barrier injury, in addition to IFN-γ / TNF-α / IL-1α and IFN-γ / TNF-α, the combination of TNF-α / IL-1α (data not shown) was included.

[0321] Table 6: Summary of TEER, permeability, and cell viability data in response to inflammatory cytokines. Abbreviation: ND (not available). TIFF2024525079000012.tif38152

[0322] In colon-derived organoid epithelial monolayers, both the IFN-γ / TNF-α / IL-1α and IFN-γ / TNF-α combinations impaired epithelial barrier integrity after 24 hours, as in previous experiments. However, treatment with the TNF-α / IL-1α combination resulted in a milder barrier impairment (a 26% reduction in TEER with TNF-α / IL-1α compared to 67% and 63% reductions in TEER with IFN-γ / TNF-α / IL-1α and IFN-γ / TNF-α, respectively), and this mild barrier impairment was not suppressed even by the highest concentration of tofacitinib (Figure 9, TNF-α / IL-1α data not shown). This result highlights the specificity of IFN-γ and tofacitinib in inducing / suppressing barrier impairment at the concentrations and time points used. Pretreatment with tofacitinib at a final concentration of 0.1 μM partially protected colonic organoid epithelial monolayers from cytokine-induced barrier damage, whereas a final concentration of 2 μM completely protected them (Figures 8A and 8B). The apparent permeability coefficient was not significantly impaired when colonic epithelial monolayers were treated with 1 ng / ml of any of the cytokine combinations used in this experiment. The apparent permeability coefficient was only slightly increased when monolayers were treated with IFN-γ / TNF-α / IL-1α and IFN-γ / TNF-α without pretreatment with tofacitinib. Cell viability measurements also mirrored the LY permeability results (Figures 8C and 8D).

[0323] Ileum-derived organoid epithelial monolayers appeared to be quite sensitive to treatment with IFN-γ / TNF-α / IL-1α and IFN-γ / TNF-α, as they completely lost their barrier integrity after 24 hours (Figure 9A and Figure 9B). However, unlike the other two cytokine combinations containing IFN-γ, treatment with TNF-α / IL-1α did not impair ileal barrier integrity (data not shown). This again emphasized the necessity of IFN-γ for barrier dysfunction, as in the colon. Ileum epithelial monolayers were protected by pretreatment with higher concentrations of tofacitinib (10 μM vs. 2 μM) compared to the colon (Figure 9A and Figure 9B). Consistent with the barrier integrity observations, LY permeability and cell viability were increased and decreased, respectively, with only the cytokine cocktail containing IFN-γ, and were again impermeable with pretreatment with 10 μM tofacitinib.

[0324] Overall, we conclude that organoid-derived epithelial monolayers were established from different GI regions on 96-well Transwells, which could be induced to different cell fates and used in barrier dysfunction induction assays for screening purposes by measuring barrier integrity, permeability, and cell viability.

[0325] Example 4. Verification of the robustness of the barrier function assay using intestinal organoid-derived monolayers Reproducibility of barrier function assays in IBD-PDO-derived epithelial monolayers IBD patient-derived organoid (IBD-PDO) monolayer cultures derived from the ileum, proximal colon, and distal colon were established following the same protocol used in previous experiments. Monolayers were pretreated with 0.1, 2, or 10 μM tofacitinib for 1 h before inducing barrier injury with 1 ng / ml of either IFN-γ / TNF-α / IL-1α, IFN-γ / TNF-α, or TNF-α / IL-1α proinflammatory cytokine combinations for 24 h. Barrier integrity of monolayers was measured at 5 and 24 h, followed by LY permeability and cell viability measurements (Figure 10, Figure 11).

[0326] The TEER value of the IBD-PDO ileal epithelial monolayer is 1000Ω / cm 2 TEER did not exceed that of IFN-γ / TNF-α / IL-1α, but changing the culture conditions to cCDM increased TEER. Epithelial monolayers showed similar sensitivity to IFN-γ / TNF-α / IL-1α and IFN-γ / TNF-α, which was inhibited by pretreatment with tofacitinib in a dose-response manner. Barrier function in IBD-PDO-derived ileal epithelial monolayers remained unchanged upon TNF-α / IL-1α treatment (data not shown), again emphasizing that IFN-γ and tofacitinib are specific in inducing and inhibiting barrier dysfunction, respectively (Figures 10A and 10B). LY permeability and cell viability experiments showed that, consistent with the barrier integrity damage experiments, LY permeability and cell viability were exacerbated by IFN-γ / TNF-α / IL-1α and IFN-γ / TNF-α, and inhibited by tofacitinib in a dose-response manner (Figures 10C and 10D).

[0327] IBD-PDO proximal colon epithelial monolayers were less sensitive to IFN-γ / TNF-α / IL-1α and IFN-γ / TNF-α, with a smaller relative decrease in cytokine-treated conditions after 5 h treatment (relative TEER values ​​of 0.59 and 0.66, compared to 0.24 and 0.29 in IBD-PDO-derived ileal epithelial monolayers and 0.11 and 0.12 in IBD-PDO-derived distal colon epithelial monolayers (data not shown). In monolayers pretreated with >0.1 μM tofacitinib, the induced damage to barrier integrity was fully restored after 24 h (data not shown). LY permeability and cell viability experiments indicated that the induced damage was not sufficient to increase monolayer permeability, and therefore the effect of tofacitinib on this readout could not be assessed. Collectively, the data suggest that IBD-PDO proximal colonic epithelial monolayers are insensitive to inflammatory cytokines and that increasing cytokine concentrations are required to overcome the dose-responsive inhibitory effects of tofacitinib.

[0328] Distal colonic epithelial monolayers from IBD-PDO were the most sensitive, with TEER values ​​decreasing to 0.11, 0.12, and 0.51, respectively, in response to IFN-γ / TNF-α / IL-1α, IFN-γ / TNF-α, and TNF-α / IL-1α, compared with 0.24, 0.29, and 0.95 in IBD-PDO ileal monolayers and 0.59, 0.66, and 0.87 in IBD-PDO proximal colonic monolayers. Treatment of monolayers with IFN-γ / TNF-α / IL-1α and IFN-γ / TNF-α led to a loss of epithelial barrier integrity after 5 h, which was compromised in the case of TNF-α / IL-1α (data not shown). Unlike other organoid monolayer cultures, the damage induced in IBD-PDO-derived distal colon monolayers was not completely inhibited at 5 h with tofacitinib at the highest concentration. The damage was reversed after 24 h, indicating that the highest concentration of tofacitinib protected the monolayer from excessive damage and provided the organoid cells with an opportunity to restore the barrier after 24 h. In response to TNF-α / IL-1α, the integrity of the barrier function was also reduced in IBD-PDO-derived distal colon monolayers, but was not inhibited or restored by tofacitinib at the highest concentration (data not shown). In LY permeability and cell viability experiments, consistent with the barrier integrity damage experiments, LY permeability and cell viability were exacerbated by IFN-γ / TNF-α / IL-1α and IFN-γ / TNF-α and were inhibited by tofacitinib in a dose-response manner (Figure 11G-L). It is worth mentioning that IBD-PDO-derived distal colon organoid cultures harbored the ATG16L1 T300A homozygous mutation, two IBD predisposing SNPs in NOD2 and IL23R. Whether the genetic susceptibility SNPs are involved in the high reactivity of the monolayer to inflammatory stimuli remains to be determined.

[0329] Collectively, these data indicate that epithelial monolayers can be generated from IBD-PDO and can be used to study barrier function along with the development of screening tools for small molecule barrier modifiers.

[0330] Example 5. Establishment of human gastrointestinal organoid epithelial monolayers Permeability and transport of various compounds are studied either by cell lines that form epithelial monolayers grown in a Transwell system or by primary intestinal epithelial tissues mounted in Ussing chambers. Many different enzymes and transporters are aberrantly expressed in adenocarcinoma cell lines such as Caco-2 cells, and Ussing chambers are very often required. An organoid-derived epithelial monolayer should combine the ease of cell lines with the precision of primary tissues. Therefore, we attempted to establish such monolayers using human duodenal and colonic organoids. This was achieved by digesting human duodenal organoids to single cells, seeding the single cells on a Transwell membrane, and differentiating them using eCDM. Similar to organoids, H&E staining of epithelial monolayer cross sections in CNMs revealed the appearance of a simple squamous epithelium, which transformed into a simple columnar epithelium after 4 days of differentiation (Figure 12A). The integrity of the epithelial monolayer was assessed by transepithelial electrical resistance (TEER). TEER was 100–200 Ω cm on days 5–6. 2 The TEER reached 1000 Ω cm and remained stable until day 9 of differentiation using the various differentiation media, after which TEER was measured for another 8 days. Among the various differentiation conditions, eCDM and gCDM achieved TEER values ​​of 1000 Ω cm 2The increase in the cellular membrane permeability of the epithelial monolayer differentiated into intestinal absorptive epithelial cells was greater than that of the cellular membrane permeability of the organoids, indicating the formation of a gap-free monolayer, which remained stable for 3 days (Fig. 12B). eCDM was selected for further experiments because it induces differentiation into intestinal absorptive epithelial cells and appropriate expression of physiologically relevant proteins such as alkaline phosphatase and mucus in organoids, resulting in a stronger barrier function of the epithelial monolayer. Next, we evaluated the paracellular permeability of the epithelial monolayer differentiated into intestinal absorptive epithelial cells on day 4 for up to 8 hours by passive diffusion of Lucifer Yellow (LY) from the apical side to the basal side and fluorescence measurement. The epithelial monolayer differentiated into intestinal absorptive epithelial cells remained impermeable for up to 4 hours (Fig. 12C). In addition to intact paracellular permeability, active transport is another important gastrointestinal epithelial function for transporting various compounds. P-glycoprotein 1 (permeability glycoprotein, Pgp1), also known as multidrug resistance protein 1 (MDR1) or ATP-binding cassette subfamily B member 1 (ABCB1), is a key protein that is widely expressed on the apical membrane of intestinal epithelium, where it transports xenobiotics (e.g., toxins or drugs) back to the lumen of the intestine. Breast cancer resistance protein (BCRP or ABCG2) is another important xenobiotic transporter expressed on the apical membrane of intestinal epithelium. The expression of these two important xenobiotic repellent transporters increases more than 10-fold after the epithelial monolayer differentiates into intestinal absorptive epithelial cells (Figure 6D). To assess the functionality of Pgp1, the Pgp1 substrate rhodamine 123 was applied to the basolateral side of the Transwell and its active transport was measured on the apical side of the epithelial monolayer. Inhibition of Pgp1 with the specific inhibitor PSC833 reduced the efflux of rhodamine 123. The results of this experiment confirm that epithelial monolayers derived from human gastrointestinal organoids have transport function, and that upon differentiation into intestinal absorptive epithelial cells, the transport function is improved in line with increased expression of transport proteins (Figures 12D and 12E). This transport function of the monolayer is Pgp1 specific, and is inhibited by PSC833 for up to 18 hours.

[0331] To further characterize human gastrointestinal epithelial monolayers, monolayers from human duodenal and colonic organoids cultured on Transwell plates were differentiated and stained to detect the expression of several key proteins (Figure 13). Similar to previous observations in 3D organoids, monolayers from duodenal and colonic organoids formed columnar epithelium upon differentiation and disappeared proliferative Ki67-positive cells (Figure 13). In H&E stained samples, differentiated colonic monolayers clearly showed more visible goblet cells than duodenal monolayers, and mucus production was also increased as confirmed by Alcian blue staining (Figure 13).

[0332] Taken together, these results indicate that organoids can be used to establish human epithelial monolayers derived from different regions of the digestive tract that can be differentiated into intestinal absorptive epithelial cells, are polarized, impermeable with barrier and transport functions, and can therefore be used to study compound permeability, metabolism, and transport.

[0333] Example 6. Polarization of human gastrointestinal organoid epithelial monolayers. As described herein, human gastrointestinal organoid-derived monolayers were seeded in eCDM, differentiated, and treated with DMSO, staurosporine, or gefitinib 3 days after seeding. Gefitinib was applied to the apical compartment, basolateral compartment, or both compartments. TEER (Figure 14A) and Lucifer Yellow permeability (Figure 14B) were measured as in the previous example.

[0334] Gefitinib is an EGFR inhibitor that suppresses proliferation. This example shows that the integrity of organoid-derived epithelial monolayers is compromised only when gefitinib is added to the basal compartment. Since EGFR is predominantly localized on the basal cell surface in human epithelial tissues, the loss of barrier integrity of the monolayer in response to basal treatment with gefitinib demonstrates that the monolayer is polarized, leak-free, and tightly adherent.

[0335] Example 7. Establishment and characterization of lung organoid monolayers. Monolayer establishment and differentiation Human lung organoids derived from three different donors (Lung-A, Lung-B, Lung-C) were passaged at high density (ratio approximately 1:2) 3–4 days before preparing monolayers. On the day of collection, organoid droplets were disrupted using well medium, and organoids were washed once with DMEM supplemented with 0.1% BSA and Pen / Strep, centrifuged at 450 × g for 5 min at 8 °C, and incubated with Mg. 2+ and Ca 2+ The organoids were digested with Accutase to single cells and small cell clumps (2-4 cells) by incubating them in a water bath and checking and resuspending the material every 5 min. Single cells were washed with Advanced DMEM / F12 supplemented with 2 mM GlutaMax, 10 mM HEPES and Pen / Strep and centrifuged twice at 450 x g for 5 min at 8 °C. Cells were passed through a pre-wetted 40 μm cell strainer and resuspended at a density of 2 million cells / ml in Lung Growth Medium (LuM) (Advanced DMEM / F12, 1% HEPES, 1% GlutaMAX, 1% penicillin / streptomycin, 1.25 mM N-acetylcysteine, 1× B27 supplement, 25 ng / ml FGF-7, 100 ng / ml FGF-10, 5 mM nicotinamide, 50 μg / ml Primocin, 250 ng / ml Rspondin-3, 500 nM SB202190 (p38i), 5 μM Y-27632 (Rho kinase inhibitor), 500 nM A83-01, 2% Noggin UPE) supplemented with 10 μM RhoKI. In parallel with the preparation of organoids, Corning® HTS Transwell® 96-well permeable supports, 0.4 μm pore size polyester membrane inserts were placed into the corresponding receiver plates. Matrigel was diluted with ice-cold PBS (Ca 2+ and Mg 2+The cells were diluted 40x with 5% PBS (containing 10% CO2). The apical surface of the Transwells was either left uncoated or coated by applying 65 μl of 2.5% Matrigel for 1 h at 37°C. After careful removal of PBS from the coated inserts, 300 μL of LuM was added to the basolateral compartment. 100 μl of cell suspension was added to the apical compartment at various cell densities (30,000-250,000 cells / Transwell) to seed the Transwells. Plates were incubated at 37°C and 5% CO2 and the medium was refreshed three times a week.

[0336] Matrigel coating was essential for the formation of lung organoid-derived cell monolayer (Figure 15A). For lung organoid-derived cell monolayer, we surprisingly found that low cell numbers (30,000 cells) seeded on Matrigel-coated Transwells resulted in higher TEER values ​​than high cell numbers (e.g., 100,000 cells or 250,000 cells) seeded on Matrigel-coated Transwells (Figure 15B). The higher TEER values ​​were obtained when low cell densities (e.g., 30,000 cells) were seeded, especially after 8 days of seeding. All cultures seeded on non-Matrigel-coated Transwells showed lower TEER values ​​than cultures seeded on Matrigel-coated Transwells. Therefore, Matrigel coating and 40,000 cells were used for all subsequent experiments on lung organoid-derived cells.

[0337] The following culture conditions were evaluated: Lung Growth Medium condition (LuM), and a condition in which the medium was changed to Lung Cilia Medium (Advanced DMEM / F12, 1% HEPES, 1% GlutaMAX, 1% penicillin / streptomycin, 1.25 mM N-acetylcysteine, 1× B27 supplement, 25 ng / ml FGF-7, 100 ng / ml FGF-10, 5 mM nicotinamide, 50 μg / ml Primocin, 250 ng / ml Rspondin-3, 500 nM SB202190 (p38i), 5 μM Y-27632 (Rho kinase inhibitor), 10 μM DAPT, 10 ng / ml BMP4) 3, 4, or 8 days after seeding (cLuM). Typically, TEER measurements of the cultures increased after changing the medium to cLuM and as the cell monolayers became more confluent.

[0338] Lung monolayers were grown in liquid-liquid interface (LLI) and air-liquid interface (ALI) formats. To determine the optimal experimental setup for lung monolayer formation, liquid-liquid interface (LLI) and air-liquid interface (ALI) cultures were evaluated. On day 13, when the monolayer was formed, ALI cultures were initiated by removing the medium from the apical compartment of the Transwell, exposing the monolayer directly to air. Cultures were maintained in this condition for 11 days until day 24. TEER values ​​were measured to monitor the integrity of the monolayer. As a control, LLI conditions were maintained in parallel by leaving the medium in both the apical and basolateral compartments for an additional 11 days until day 24. TEER values ​​were measured to monitor the integrity of the monolayer.

[0339] Four or eight days after changing the cell culture medium to cLuM, the morphology, barrier function, marker expression (this example), and transport function (example 8) of the pulmonary organoid-derived monolayer were evaluated.

[0340] form Morphology of lung organoid monolayers was assessed using H&E staining and revealed a monolayer of pseudostratified epithelial cells during both proliferation (in LuM medium) and differentiation (in cLuM medium) (Figure 16A-C). Lung organoid monolayers exhibit heterogeneous cell populations, which is reflected in their barrier properties (TEER and permeability, see below) and histological appearance. Small "air bubbles" were observed in the cell layer medium, which correlated with the expression of alveolar markers (Figure 16A cLuM ALI, Figure 17A LuM LLI and ALI), suggesting that lung organoid monolayers are a useful lung epithelial model.

[0341] transparency The permeability of the monolayers was assessed by measuring TEER throughout the experiment (Figures 17A-C). TEER values ​​of the lung monolayers increased after seeding and remained measurable in the different culture media (cLuM or LuM) and culture formats (ALI or LLI).

[0342] Permeability was also assessed by Lucifer Yellow assay (Figure 18A). Briefly, 60 μM Lucifer Yellow was added to the apical compartment. After 60 min of incubation at 37°C, the diffusion of Lucifer Yellow to the basolateral compartment was measured. The results are shown in Figures 18B-D. Overall, lung monolayer cultures showed less permeability to Lucifer Yellow than control samples (blanks).

[0343] Marker Expression The expression of various pulmonary markers and transport proteins in lung monolayers was measured using RT-qPCR. Expression was also evaluated in the organoids used to seed the monolayers. Figure 19A and Figure 19B show the expression levels of KRT5 and SPDEF in organoid monolayers. Figure 19C and Figure 19D show the expression levels of KRT5 and SPDEF in lung organoids. Figure 19E and Figure 19F show the expression levels of FOXJ1 and SFTPA1 in organoid monolayers. Figure 19G shows the expression levels of FOXJ1 in lung organoids. Figure 19H and Figure 19I show the expression levels of OCTN1 and MRP1 in organoid monolayers. Figure 19J and Figure 19K show the expression levels of OCTN1 and MRP1 in lung organoids.

[0344] Lung organoid monolayers were grown in LuM or differentiated in cLuM for 4 or 8 days. Lung organoids were cultured in LuM or cLuM for various periods as described. The expression of the following lung markers was evaluated: KRT5 (lung basal cell marker), SPDEF (goblet cell marker), FOXJ1 (ciliated cell marker), and SFTPA1 (alveolar marker). The expression of transport proteins OCTN1 and MRP1 was also measured. The results are shown in Figure 19.

[0345] The lung markers KRT5 and SPDEF were detected in both lung monolayers and lung organoids. The ciliated cell marker FOXJ1 was detected in one of the lung monolayers and two of the lung organoids. The alveolar marker SFTPA1 was detected in the lung-B culture sample (Figure 19F), which correlated with the appearance of "bubble" structures visible when the lung monolayers were cultured in LuM medium in both LLI and ALI formats (Figure 16B). The lung-C culture sample showed the most cilia in the histological images (Figure 16C), and the ciliated cell marker FOXJ1 was highly expressed in this culture (Figure 19E). The lung-C culture sample also showed the highest expression of the OCTN1 transporter. The expression of the transporter OCTN1 was detected in both lung monolayers and lung organoids, and the expression of the transporter MRP1 was detected in all lung monolayer and lung organoid samples in all conditions.

[0346] Example 8. Calcein transport assay in lung organoid monolayers. This example demonstrates the development of a transport assay that can measure the transporter function of pulmonary organoid monolayers through the accumulation of fluorescent dye in monolayers.Lung-C pulmonary monolayers were selected for transport assay because the barrier function of Lung-C pulmonary monolayers was robust (Figure 17C and Figure 18D).

[0347] Calcein transport assay-accumulation assay Calcein transport from the basolateral compartment into lung monolayers grown in either LuM LLI or LuM ALI conditions as described in Example 7 was measured 16 days after seeding. For ALI cultures, cells were transferred to the ALI culture format 4 days after seeding. Cells were cultured in the "accumulation" assay format with an MRP1 transporter specific inhibitor (MK571) and a P-gp specific inhibitor (PSC833) as follows (Figures 20A-C). 1. Remove the medium and rinse the cells with PBS. 2. Add fresh buffer to both the apical and basolateral compartments in one of the following conditions (37° C.) according to the expected expression of the MRP transporter: 1.10 μM MK571, 30 min, 2.5 μM MK571, 30 min, 3.1 μM PSC-833, 30 min, or 4. Untreated control, 30 min. 3. Add Calcein AM to the basolateral compartment at a final concentration of 250 nM and incubate at 37° C. for 30 minutes. 4. Analyze the intracellular accumulation of Calcein AM by fluorescence emission (excitation λ 490 nm, emission λ 520 nm). 5. Assess the differences in accumulation.

[0348] In both LuM LLI and LuM ALI cultures, increased intracellular accumulation of calcein AM was observed in the presence of both MRP1 and P-gp inhibitors (FIG. 20D).

[0349] Calcein transport assay - pulse-chase assay Calcein AM transport assays were performed under similar conditions as the "accumulation" format with Lung-C monolayer cultures, with the following modification: monolayers were exposed to 250 nM calcein AM for 30 min at 30°C. After washing with PBS, baseline intracellular fluorescence was measured (T=0). Monolayers were then further incubated for an additional 2 h at 37°C with or without inhibitors (MK571 or PSC-833) in PBS. Intracellular fluorescence and apical and basolateral medium fluorescence were measured at T=2 h (Figure 21A).

[0350] In both LLI and ALI formats, inhibition of MRP1 increases intracellular accumulation of calcein AM (Figures 21B and 21C). After calcein AM is removed from the medium of the apical and / or basolateral compartments, inhibition of MRP1 reduces calcein AM efflux in both LLI and ALI formats (Figures 21D and 21E). This is in line with the consistent expression of MRP1 across the various culture conditions. Inhibition of P-gp had no significant effect on calcein AM accumulation or efflux.

[0351] Example 9. Establishment and characterization of kidney organoid monolayers. Monolayer establishment and differentiation Human kidney organoids derived from three different donors (Kidney-A, Kidney-B, and Kidney-C) were passaged at high density (ratio approximately 1:2) 3-4 days before preparing monolayers. On the day of collection, organoid droplets were disrupted using well medium, and organoids were washed once with DMEM supplemented with 0.1% BSA and Pen / Strep, centrifuged at 450 × g for 5 min at 8 °C, and incubated with Mg. 2+ and Ca 2+The organoids were digested with Accutase to single cells and small cell clumps (2-4 cells) by incubating them in a water bath and checking and resuspending the material every 5 min. Single cells were washed with Advanced DMEM / F12 supplemented with 2 mM GlutaMax, 10 mM HEPES and Pen / Strep and centrifuged twice at 450 x g for 5 min at 8 °C. Cells were passed through a pre-wetted 40 μm cell strainer and resuspended at a density of 2 million cells / ml in kidney growth medium (ADMEM / F12, 1% HEPES, 1% GlutaMAX, 1% penicillin / streptomycin, 1.5% B27 supplement, 10% Rspo1 conditioned medium, 50 ng / ml EGF, 100 ng / ml FGF-10, 10 μM Rho-kinase inhibitor Y-27632, 5 μM A8301, 0.1 mg / ml Primocin) supplemented with 10 μM RhoKI. In parallel with the preparation of organoids, Corning® HTS Transwell® 96-well permeable supports, polyester membrane inserts with 0.4 μm pore size, were placed into the corresponding receiver plate. Matrigel was resuspended in ice-cold PBS (Ca 2+ and Mg 2+ The cells were diluted 40x in 100 mM NaCl (containing 0.1% CO2). The apical surface of the Transwells was either left uncoated or coated by applying 65 μl of 2.5% Matrigel for 1 h at 37°C. After careful removal of the PBS from the coated inserts, 300 μl of kidney growth medium was added to the basolateral compartment. The Transwells were seeded by adding 100 μl of cell suspension to the apical compartment at various cell densities (30,000-250,000 cells / Transwell). The plates were incubated at 37°C and 5% CO2 and the medium was refreshed three times a week.

[0352] Coating the Transwells with Matrigel and seeding higher numbers of cells resulted in higher TEER values ​​in the monolayers (Figure 22B), with no additional benefit seen with seeding densities above 100,000 cells / Transwell (Figure 22A). Therefore, Matrigel coating and 100,000 cells / Transwell were used for all subsequent experiments.

[0353] The following culture conditions were evaluated: consistent culture in kidney growth medium (KEM), addition of 1 uM decitabine to kidney growth medium 2 days after seeding (DAC), and changing the medium to kidney differentiation medium (ADMEM / F12, 1% HEPES, 1% GlutaMAX, 1% penicillin / streptomycin) 3 days after seeding (KDM). Kidney growth medium and kidney differentiation medium have been previously described by Schutgens et al. (Nature Biotechnology 37:303-313, 2019). Decitabine is a DNA methyltransferase inhibitor, and the inventors hypothesized that its addition may enhance the expression of transport proteins.

[0354] Seven days after seeding, kidney monolayers were assessed for morphology, barrier function, marker expression (this example) and transport function (example 10).

[0355] form Morphology of kidney organoid monolayers assessed by H&E staining revealed that the cell layer was very thin and contained a mixture of various cell types (Figure 23A-C), and kidney-C-derived monolayers grown in KDM contained ciliated cells (Figure 23C).

[0356] transparency The permeability of the monolayer was assessed by measuring the TEER throughout the experiment (FIGS. 24A-C).

[0357] Permeability was also assessed by Lucifer Yellow assay. Briefly, 60 μM Lucifer Yellow was added to the apical compartment. After incubation at 37° C. for 60 min, the diffusion of Lucifer Yellow to the basolateral compartment was measured. The results are shown in FIG. 25A-C.

[0358] Marker Expression The expression of various renal markers and transport proteins in the monolayers was measured using RT-qPCR. Expression was also evaluated in the organoids used to seed the monolayers. Organoids were grown in KEM or differentiated in KDM for 4 or 8 days. The expression of the following renal markers was evaluated: ABCC4 (proximal tubule marker), PAX8 (renal epithelium marker), CLDN10 (Henle's loop marker), SLC12A3 (distal tubule marker), and AQP3 (collecting duct marker). The expression of transport proteins OAT1, OAT3, OCT2, MATE1, and MATE2-K was also measured. The results are shown in Figure 26.

[0359] The expression level of SLC12A3 was below the threshold in organoids and organoid-derived monolayers (not shown). The same was true for AQP3 in organoids (not shown), but expression was detected in one of the monolayers (Figure 26G). Expression of OAT1 and OAT3 was not detected in either organoids or organoid-derived monolayers (not shown).

[0360] In summary, kidney monolayers were found to display heterogeneity in cellular composition, which was reflected in their barrier properties as shown using TEER and Lucifer Yellow assays (Figures 24 and 25). The same renal markers expressed in organoids were expressed at similar levels in monolayers, with the exception of CLDN10, which was more highly expressed in kidney-A monolayers compared to kidney-A organoids. Monolayers also expressed three of the five transporters evaluated, which are also expressed in organoids, but at different levels. No further increase in transporter expression was observed in KDM monolayers.

[0361] Example 10. Transport assay in renal organoid monolayers. This example demonstrates the development of a transport assay that can measure the transporter function of kidney organoid monolayer membranes through the accumulation of fluorescent dye in the epithelium.Kidney-C organoid line was selected for transport assay because the barrier function of kidney-C organoid line was robust (Figure 24C and Figure 25C).

[0362] Calcein transport assay Transport of calcein from the basolateral compartment into kidney monolayers grown in either KEM or DAC conditions as described in Example 9 was measured 7 days after plating in the presence or absence of the P-gp inhibitor PSC-833 as follows: 6. Remove the medium and rinse the cells with PBS. 7. Add fresh buffer at one of the following conditions (37° C.): 1.1 μM PSC-833, 30 min, or 2. Untreated control, 30 min. 8. Add Calcein AM to the basolateral compartment at a final concentration of 250 nM and incubate at 37° C. for 30 minutes. 9. Analyze intracellular accumulation of Calcein AM by fluorescence emission (excitation λ 490 nm, emission λ 520 nm). 10. Assess the differences in accumulation. In the presence of P-gp inhibitors, increased intracellular accumulation of calcein AM was observed (FIG. 27).

[0363] Rhodamine transport assay Transport of rhodamine from the basolateral compartment into kidney monolayers grown in either KEM or DAC conditions as described in Example 9 was measured 7 days after plating in the presence or absence of the P-gp inhibitor PSC-833 and / or the OCT2 inhibitor decinium-22 as follows: 1. Remove the medium and rinse the cells with PBS. 2. Add fresh buffer at one of the following conditions (37° C.): 1.1 μM Decinium-22, 30 min, 2.1 μM PSC-833, 30 min, 3.1 μM Decinium-22 and 1 μM PSC-833, 30 min, or 4. Untreated control, 30 min. 3. Add Rhodamine 123 to a final concentration of 250 nM to the basolateral compartment and incubate at 37° C. for 30 minutes. 4. The intracellular accumulation of Rhodamine 123 is analyzed by fluorescence emission (excitation λ 490 nm, emission λ 520 nm). 5. Assess the differences in accumulation.

[0364] In the presence of P-gp inhibitors, increased intracellular accumulation of rhodamine was observed, whereas decreased accumulation was observed with OCT2 inhibitors. DAC treatment did not increase the loading / transport of rhodamine 123 (Figure 28).

Claims

1. A method for obtaining intestinal organoid-derived monolayers, comprising the steps of: i. digesting or dissociating one or more intestinal organoids into a suspension of single cells and / or organoid fragments; ii. Seeding a semipermeable membrane with the suspension; iii. Culturing the cells and / or organoid fragments in the presence of a growth medium until a monolayer is formed; and iv. Culturing the monolayer in the presence of a differentiation medium comprising a Notch inhibitor, an EGFR pathway inhibitor and a Wnt agonist.

2. (i) the monolayer has a transepithelial electrical resistance (TEER) of about 100 Ω cm ２ is cultured in the presence of growth medium until (ii) the TEER of the monolayer is further increased during the step of culturing the monolayer in the presence of the differentiation medium, and optionally, the TEER of the monolayer is increased to 500 Ω cm during the step of culturing the monolayer in the presence of the differentiation medium. ２ Super, 600Ω・cm ２ Super, 700Ω・cm ２ Super, 800Ω・cm ２ Super, 900Ω・cm ２ Super, 1000Ω・cm ２ Super, 1100Ω・cm ２ Super, 1200Ω・cm ２ Super, 1300Ω・cm ２ Super, 1400Ω・cm ２ or greater than 1500 Ω·cm ２ reach super; (iii) the growth medium comprises a receptor tyrosine kinase ligand, a BMP inhibitor and a Wnt agonist, and optionally nicotinamide and a p38 MAPK inhibitor such as SB202190, and further optionally (a) the receptor tyrosine kinase ligand is a ligand for RTK class I (EGF receptor family) (ErbB family), a ligand for RTK class II (insulin receptor family), a ligand for RTK class IV (FGF receptor family), or a ligand for RTK class VI (HGF receptor family), and further optionally, the receptor tyrosine kinase ligand is selected from the group consisting of epidermal growth factor (EGF), neuregulin, fibroblast growth factor (FGF), hepatocyte growth factor (HGF), and insulin-like growth factor (IGF); and / or (b) the BMP inhibitor is selected from the group consisting of noggin, sclerostin, chordin, CTGF, follistatin, gremlin, tsg, sog, LDN193189, or dorsomorphin; (iv) the Wnt agonist is selected from the group consisting of Rspondin, Wnt-conditioned medium, and Wnt substitute; (v) the Notch inhibitor is a gamma secretase inhibitor, optionally selected from the group consisting of DAPT, dibenzazepine (DBZ), benzodiazepine (BZ), and LY-411575; (vi) the EGFR pathway inhibitor is selected from (1) an EGFR inhibitor such as gefitinib, (2) an EGFR and ErbB2 inhibitor such as afatinib, (3) an inhibitor of the RAS-RAF-MAPK pathway, (4) an inhibitor of the PI3K / AKT pathway, and (5) an inhibitor of the JAK / STAT pathway, optionally wherein the EGFR pathway inhibitor is an inhibitor of the RAS-RAF-MAPK pathway, e.g., a MEK inhibitor such as PD0325901; and / or (vii) the monolayer film is (a) cultured in the presence of growth medium for at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, or at least 10 days, preferably the monolayer is cultured in the presence of growth medium for 3-9 days; (b) cultured in the presence of said differentiation medium for at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days or more, preferably said monolayer is cultured in the presence of differentiation medium for 4-8 days; and / or (c) cultured in the presence of an extracellular matrix; The method of claim 1.

3. A monolayer membrane derived from intestinal organoids obtainable or obtained by the method of claim 1.

4. (i) the monolayer is composed of the following cell types: Lgr5 ＋ Stem cells, intestinal absorptive epithelial cells, goblet cells, Paneth cells and enteroendocrine cells and / or (ii) the monolayer is derived from a mammal, optionally the monolayer is derived from a human, and further optionally the human has a disease or disorder of the digestive system, such as inflammatory bowel disease (e.g., Crohn's disease or ulcerative colitis), celiac disease or leaky gut syndrome; The method according to claim 1 or 2. (i) The monolayer is a cell of the following cell type: Lgr5 + stem cells, intestinal absorptive epithelial cells, goblet cells, Paneth cells, and enteroendocrine cells and / or (ii) the monolayer is derived from a mammal, optionally the monolayer is derived from a human, and further optionally the human has a disease or disorder of the digestive system, such as inflammatory bowel disease (e.g., Crohn's disease or ulcerative colitis), celiac disease or leaky gut syndrome; The organoid-derived monolayer membrane of claim 3.

6. A method for obtaining a lung organoid-derived monolayer, comprising the steps of: i. digesting or dissociating one or more lung organoids into a suspension of single cells and / or organoid fragments; ii. seeding a semipermeable membrane with the suspension; and iii. Culturing the cells and / or organoid fragments in the presence of a growth medium until a monolayer is formed.

7. (i) iv. further comprising culturing the monolayer in the presence of a differentiation medium; (ii) the monolayer is cultured in the presence of an extracellular matrix; (iii) the growth medium comprises one or more receptor tyrosine kinase ligands, Wnt agonists, TGF-beta inhibitors, BMP inhibitors, and optionally a Rho kinase inhibitor such as Y-27632, and / or a p38 MAPK inhibitor such as SB202190, and further optionally (a) the receptor tyrosine kinase ligand is a ligand for RTK class I (EGF receptor family) (ErbB family), a ligand for RTK class II (insulin receptor family), a ligand for RTK class IV (FGF receptor family), or a ligand for RTK class VI (HGF receptor family), and further optionally, the receptor tyrosine kinase ligand is selected from the group consisting of epidermal growth factor (EGF), neuregulin, fibroblast growth factor (FGF), hepatocyte growth factor (HGF), and insulin-like growth factor (IGF); (b) the BMP inhibitor is selected from the group consisting of noggin, sclerostin, chordin, CTGF, follistatin, gremlin, tsg, sog, LDN193189, or dorsomorphin; (c) the Wnt agonist is selected from the group consisting of Rspondin, Wnt-conditioned medium, and Wnt-surrogate; and / or (d) the TGF-beta inhibitor is selected from the group consisting of A83-01, SB-431542, SB-505124, SB-525334, LY 364947, SD-208 and SJN 2511; (iv) the differentiation medium comprises one or more receptor tyrosine kinase ligands, Wnt agonists, Notch inhibitors, BMP pathway activators, and optionally a Rho kinase inhibitor such as Y-27632, and / or a p38 MAPK inhibitor such as SB202190, and further optionally (a) the receptor tyrosine kinase ligand is a ligand for RTK class I (EGF receptor family) (ErbB family), a ligand for RTK class II (insulin receptor family), a ligand for RTK class IV (FGF receptor family), or a ligand for RTK class VI (HGF receptor family), and further optionally, the receptor tyrosine kinase ligand is selected from the group consisting of epidermal growth factor (EGF), neuregulin, fibroblast growth factor (FGF), hepatocyte growth factor (HGF), and insulin-like growth factor (IGF); (b) the Wnt agonist is selected from the group consisting of Rspondin, Wnt conditioned medium, and Wnt substitute; (c) the Notch inhibitor is a gamma secretase inhibitor, optionally a gamma secretase inhibitor selected from the group consisting of DAPT, dibenzazepine (DBZ), benzodiazepine (BZ), and LY-411575; and / or (d) the BMP pathway activator is selected from the group consisting of BMP7, BMP4, and BMP2; (v) seeding the semi-permeable membrane with less than about 20,000 cells, less than about 30,000 cells, less than about 40,000 cells, less than about 50,000 cells, less than about 60,000 cells, less than about 70,000 cells, less than about 80,000 cells, less than about 90,000 cells, less than about 100,000 cells, or less than about 250,000 cells, e.g., in a standard 96-well format; (vi) seeding the semi-permeable membrane with about 30,000 cells, about 40,000 cells, about 50,000 cells, about 60,000 cells, about 70,000 cells, about 80,000 cells, or about 90,000 cells, e.g., in a standard 96-well format; (vii) seeding the semi-permeable membrane with about 5,000 to 500,000 cells, about 10,000 to 250,000 cells, about 20,000 to 100,000 cells, about 30,000 to 50,000 cells, about 35,000 to 45,000 cells, or preferably about 40,000 cells, e.g., in a standard 96-well format; (viii) the suspension of single cells and / or organoid fragments is adjusted to about 0.2 x 10 per mL prior to seeding. ６ Less than cells, approximately 0.3 x 10 per mL ６ Less than cells, approximately 0.4 x 10 per mL ６ Less than cells, approximately 0.5 x 10 per mL ６ Less than a cell, approximately 10 per mL ６ Less than 2 x 10 cells per mL ６ Less than 3 x 10 cells per mL ６ Less than 4 x 10 cells per mL ６ Less than or equal to approximately 5 x 10 cells per mL ６ including subcellular conditioning; (ix) the suspension of single cells and / or organoid fragments is diluted to about 0.2 x 10 per mL prior to seeding. ６ Cells, approximately 0.3 x 10 per mL ６ Cells, approximately 0.4 x 10 per mL ６ Cells, approximately 0.5 x 10 per mL ６ Cells, approximately 10 per mL ６ Cells, approximately 2 x 10 per mL ６ Cells, approximately 3 x 10 per mL ６ Cells, approximately 4 x 10 per mL ６ Approximately 5 x 10 cells or per mL ６ conditioning the cells; (x) the suspension of single cells and / or organoid fragments is diluted to about 0.1 to 1 x 10 per mL prior to seeding. ６ Cells, approximately 0.25-0.75 x 10 per mL ６ Cells, approximately 0.3-0.5 x 10 per mL ６ Cells, approximately 0.35-0.45 x 10 per mL ６ Cells, preferably about 0.4 x 10 per mL ６ conditioning the cells; (xi) the monolayer film is (a) cultured in the presence of growth medium for at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days or more, preferably the monolayer is cultured in the presence of growth medium for 3-8 days; and / or (b) cultured in the presence of differentiation medium for at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days or more, preferably the monolayer is cultured in the presence of differentiation medium for 8 days; and / or (xii) further comprising the step of removing said proliferation medium or said differentiation medium from the apical compartment, optionally wherein said medium is removed from the apical compartment 10 to 16 days after seeding, for example 11, 12, 13, 14 or 15 days after seeding, preferably 13 days after seeding; The method of claim 6.

8. A pulmonary organoid-derived monolayer membrane obtained or obtainable by the method of claim 6.

9. (i) the monolayer is composed of the following cell types: Club cells, basal cells, ciliated cells, goblet cells, type I pneumocytes and type II pneumocytes and / or (ii) the monolayer is derived from a mammal, e.g., a human; 8. The method according to claim 6 or 7. (i) The monolayer is a cell of the following cell type: Club cells, basal cells, ciliated cells, goblet cells, type I pneumocytes and type II pneumocytes and / or (ii) the monolayer is derived from a mammal, e.g., a human; The organoid-derived monolayer membrane of claim 8.

11. A method for obtaining a kidney organoid-derived monolayer, comprising the steps of: i. digesting or dissociating one or more kidney organoids into a suspension of single cells and / or organoid fragments; ii. seeding a semipermeable membrane with the suspension; and iii. Culturing the cells and / or organoid fragments in the presence of a growth medium until a monolayer is formed.

12. (i) iv. further comprising culturing the monolayer in the presence of a differentiation medium; (ii) the monolayer is cultured in the presence of an extracellular matrix; (iii) the growth medium comprises one or more receptor tyrosine kinase ligands, Wnt agonists, and TGF-beta inhibitors, and optionally a Rho kinase inhibitor; (iv) seeding the semi-permeable membrane with less than about 100,000 cells, less than about 150,000 cells, less than about 200,000 cells, or less than about 250,000 cells, e.g., in a standard 96-well format; (v) seeding the semi-permeable membrane with about 30,000 cells, about 40,000 cells, about 50,000 cells, about 60,000 cells, about 70,000 cells, about 80,000 cells, about 90,000 cells, or about 100,000 cells, e.g., in a standard 96-well format; (vi) seeding the semi-permeable membrane with about 20,000 to 500,000 cells, about 30,000 to 400,000 cells, about 40,000 to 300,000 cells, about 50,000 to 250,000 cells, about 60,000 to 200,000 cells, about 70,000 to 150,000 cells, about 80,000 to 120,000 cells, or preferably about 100,000 cells, e.g., in a standard 96-well format; (vii) the suspension of single cells and / or organoid fragments is adjusted to about 0.5 x 10 per mL prior to seeding. ６ Less than 0.6 x 10 cells per mL ６ Less than cells, approximately 0.7 x 10 per mL ６ Less than 0.8 x 10 cells per mL ６ Less than cells, approximately 0.9 x 10 per mL ６ Less than a cell, approximately 10 per mL ６ Less than 1.1 x 10 cells per mL ６ Less than 1.2 x 10 cells per mL ６ Less than 1.3 x 10 cells per mL ６ Less than 1.4 x 10 cells per mL ６ Less than cells, or approximately 1.5 x 10 per mL ６ including subcellular conditioning; (viii) the suspension of single cells and / or organoid fragments is adjusted to about 0.2 x 10 per mL prior to seeding. ６ Cells, approximately 0.3 x 10 per mL ６ Cells, approximately 0.4 x 10 per mL ６ Cells, approximately 0.5 x 10 per mL ６ Cells, approximately 10 per mL ６ Cells, approximately 1.5 x 10 per mL ６ Cells, approximately 2 x 10 per mL ６ Cells, approximately 3 x 10 per mL ６ Cells, approximately 4 x 10 per mL ６ cells, or approximately 5 x 10 per mL ６ Cells, preferably about 10 per mL ６ conditioning the cells; (ix) The suspension of single cells and / or organoid fragments is diluted to about 0.1 to 5 x 10 per mL prior to seeding. ６ Cells, approximately 0.25-2.5 x 10 per mL ６ Cells, approximately 0.5-1.5 x 10 per mL ６ Cells, approximately 0.75-1.25 x 10 per mL ６ Cells, approximately 0.8-1.2 x 10 per mL ６ Cells, preferably about 10 per mL ６ conditioning the cells; (x) the monolayer film is (a) cultured in the presence of growth medium for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days or more, preferably the monolayer is cultured in the presence of growth medium for 1-3 days, more preferably for 2 days; and / or (b) cultured in the presence of differentiation medium for at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days or more, preferably the monolayer is cultured in the presence of differentiation medium for 3-5 days, more preferably for 4 days; and / or (xi) further comprising the step of adding a histone deacetylase inhibitor, such as decitabine, to the proliferation medium or the differentiation medium, optionally wherein the histone deacetylase inhibitor is added 1 to 3 days after seeding, preferably 2 days after seeding; The method of claim 11.

13. A renal organoid-derived monolayer obtained or obtainable by the method of claim 11.

14. 25 Ω cm ２ Super, 50Ω・cm ２ Super, 75Ω・cm ２ Super, 100Ω・cm ２ Super, 200Ω・cm ２ Super, 300Ω・cm ２ Super, 400Ω・cm ２ Super, 500Ω・cm ２ Super, 600Ω・cm ２ Super, 700Ω・cm ２ Super, 800Ω・cm ２ Super, 900Ω・cm ２ Super, 1000Ω・cm ２ Super, 1100Ω・cm ２ Super, 1200Ω・cm ２ Super, 1300Ω・cm ２ or greater than 1400 Ω·cm ２ The renal organoid-derived monolayer membrane of claim 13, having a TEER of greater than 1000kJ / cm.

15. (i) the monolayer is composed of the following cell types: Proximal tubule cells, renal epithelial cells, Henle's loop cells, distal tubule cells and collecting duct cells and / or (ii) the monolayer is derived from a mammal, e.g., a human; 13. The method of claim 11 or 12. (i) The monolayer is a cell of the following cell type: Proximal tubule cells, renal epithelial cells, Henle's loop cells, distal tubule cells and collecting duct cells and / or (ii) the monolayer is derived from a mammal, e.g., a human; 15. An organoid-derived monolayer membrane according to claim 13 or 14.

17. Use of an organoid-derived monolayer membrane described in any one of claims 3, 8 and 13 in an assay to evaluate epithelial viability, metabolic activity, permeability, barrier function integrity and / or transport protein activity.

18. 1. A method for identifying a compound capable of modulating epithelial viability, metabolic activity, permeability, barrier integrity and / or transport protein activity, comprising the steps of: i. Contacting an organoid-derived monolayer, such as the organoid-derived monolayer of any one of claims 3, 8 and 13, with one or more candidate molecules; ii. Assessing the viability, metabolic activity, permeability and / or barrier function integrity of the organoid-derived monolayer and / or the activity of transport proteins in the organoid-derived monolayer.

19. 1. A method for assessing the effect of a compound on epithelial viability, metabolic activity, permeability, barrier integrity and / or transport protein activity, comprising: i. Contacting an organoid-derived monolayer, such as the organoid-derived monolayer of any one of claims 3, 8 and 13, with said compound; ii. Assessing the viability, metabolic activity, permeability and / or barrier function integrity of the organoid-derived monolayer and / or the activity of transport proteins in the organoid-derived monolayer.

20. 19. The method of claim 18, further comprising contacting said organoid-derived monolayer with one or more inflammatory cytokines, optionally wherein said one or more inflammatory cytokines are selected from the group consisting of IFN-γ, TNF-α, and IL-1α.

21. 1. A method for identifying mutations associated with epithelial viability, metabolic activity, permeability, barrier function integrity and / or transport protein activity, comprising the steps of: i. assessing the viability, metabolic activity, permeability and / or barrier function integrity of an organoid-derived monolayer, e.g., an organoid monolayer according to any one of claims 3, 8 and 13, and / or the activity of transport proteins in the organoid-derived monolayer; ii. Determining the presence of one or more mutations in the genome of one or more cells in the organoid-derived monolayer.

22. i. Obtaining an organoid-derived monolayer from the human subject as described in any one of claims 1, 6 and 11; and ii. Testing the viability, metabolic activity, permeability and / or barrier function integrity of said organoid-derived monolayer and / or the activity of transport proteins in said organoid-derived monolayer.

1. A method for diagnosing a disease or affliction affecting epithelial viability, metabolic activity, permeability, barrier function integrity and / or transport protein activity in a human subject, or determining an increased risk of said disease or affliction, comprising:

23. (i) the reference value is a value obtained from a control, e.g., an organoid-derived monolayer obtained from a healthy human subject; and / or (ii) the disease or affliction is a disease or disorder of the digestive system, such as inflammatory bowel disease (e.g., Crohn's disease or ulcerative colitis), celiac disease or leaky gut syndrome; 23. The method of claim 22.

24. A method for predicting the likelihood of a patient responding to a candidate compound, comprising: i. Obtaining an organoid-derived monolayer from the patient as described in any one of claims 1, 6 and 11; ii. contacting the organoid-derived monolayer with the compound; and iii. Assessing the viability, metabolic activity, permeability and / or barrier function integrity of the organoid-derived monolayer and / or the activity of transport proteins in the organoid-derived monolayer.

25. (i) assessing the integrity of the barrier function of the organoid-derived monolayer comprises measuring the TEER of the organoid-derived monolayer; (ii) assessing the permeability of the organoid-derived monolayer comprises measuring the passive diffusivity of a reporter compound across the monolayer, and optionally, the reporter compound is a dye, optionally a fluorescent dye such as Lucifer Yellow; (iii) assessing the activity of the transport protein comprises measuring the rate of transport of a substrate of the transport protein across the monolayer, optionally in the presence of an inhibitor of the transport protein, wherein optionally the substrate is a dye such as rhodamine 123 or calcein AM; and / or (iv) assessing the activity of the transport protein comprises measuring the rate of transport of a substrate of the transport protein into cells of the monolayer, optionally in the presence of an inhibitor of the transport protein, and optionally the substrate is a dye such as rhodamine 123 or calcein AM.

20. The method of claim 18.

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