Culture medium for expanding mammary epithelial stem cells

Abstract

Improved culture methods for expanding epithelial stem cells and for obtaining organoids, culture media involved in said methods, and uses of said organoids are provided. [Solution] A method for expanding mammary epithelial stem cells includes the steps of: preparing a mammary stem cell population; preparing a culture medium containing an ErbB3 / 4 ligand, one or more FGFR2b ligands, and a Wnt agonist; contacting the stem cells with the culture medium; and culturing the cells under appropriate conditions.

Description

[Technical field]

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

[0002] Technical Field The present invention is in the field of mammary epithelial stem cell culture media and methods, in particular culture media and methods for expanding populations of mammary epithelial stem cells, eg, human mammary epithelial stem cells. [Background technology]

[0003] background There is a high interest in culture media and methods for expanding stem cell populations. Stem cell populations have many applications. For example, stem cells and their differentiated progeny can be used in cell assays, drug screening, and toxicity assays. Stem cells also show potential for cell therapy, for example, in regenerative medicine to treat damaged tissue. In addition, efficient cell culture media is important to provide and maintain cell populations for research purposes.

[0004] Methods have been described for the long-term culture of epithelial stem cells or tissue fragments derived from several tissues (e.g., pancreas, colon, intestinal crypts, stomach, liver, and prostate) (see WO2010 / 090513 (Patent Document 1), WO2012 / 014076 (Patent Document 2), and WO2012 / 168930 (Patent Document 3)). However, these methods have been found to be unable to support the long-term culture of epithelial stem cells or tissue fragments derived from breast tissue.

[0005] A culture system for culturing epithelial stem cells derived from normal breast tissue is described in Pasic et al. (2011) Genes & Development 25: 1641-1653 (Non-Patent Document 1). However, this system cannot support long-term culture of breast organoids. It has been observed that cultures using this system stop growing after 2-3 passages. Therefore, it is not possible to obtain a large number of stem cells using this technique.

[0006] A culture system for culturing circulating tumor cells derived from breast cancer patients is described in Yu et al. (2014) Science 345(6193): 216-220. However, the authors of this paper did not provide instructions for culturing mammary epithelial stem cells obtained from normal breast tissue, primary tumors, or metastases. Furthermore, circulating breast tumor cells cultured using the Yu et al. system form cell aggregates.

[0007] Therefore, there is a need for the culture medium and method for culturing the breast epithelial stem cells obtained from normal breast tissue, primary tumor or metastasis, which allows long-term culture.There is also a need for the culture medium and method for culturing circulating breast tumor cells, which allows the formation of breast organoids that closely resemble breast tumors. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] WO2010 / 090513 [Patent Document 2] WO2012 / 014076 [Patent Document 3] WO2012 / 168930 [Non-patent literature]

[0009] [Non-Patent Document 1] Pasic et al. (2011) Genes & Development 25: 1641-1653 [Non-Patent Document 2] Yu et al. (2014) Science 345(6193): 216-220 Summary of the Invention

[0010] The present invention provides a method for expanding mammary epithelial stem cells, comprising the steps of: Providing a mammary stem cell population; providing a culture medium comprising an ErbB3 / 4 ligand, one or more FGFR2b ligands, and a Wnt agonist; contacting the stem cells with a culture medium; and Culturing the cells under appropriate conditions.

[0011] Additionally, the present invention provides a culture medium comprising an ErbB3 / 4 ligand, and one or more FGFR2b ligands, and a Wnt agonist.

[0012] Additionally, the present invention provides a culture medium comprising an ErbB3 / 4 ligand suitable for expanding a population of mammary epithelial stem cells for at least four passages.

[0013] Furthermore, the present invention provides breast organoids comprising a population of breast epithelial stem cells. In some embodiments, said breast organoids are obtainable or obtained by the method of the present invention.

[0014] Furthermore, the present invention provides a mammary organoid of the present invention in a culture medium of the present invention.

[0015] Furthermore, the present invention provides the use of the breast organoids of the present invention or cells derived from said organoids in drug discovery screening; toxicity assays; investigating histogenesis, cell lineages, or differentiation pathways; studying gene expression, including recombinant gene expression; investigating mechanisms involved in tissue damage or repair; investigating inflammatory and infectious diseases; studying mechanisms of pathogenesis; or studying mechanisms of cell transformation or the pathogenesis of cancer.

[0016] Furthermore, the present invention provides the organoids of the invention or cells derived from said organoids for use in medicine.

[0017] Furthermore, the present invention provides a culture medium comprising a p53 stabilizer. [The present invention 1001] 1. A method for expanding mammary epithelial stem cells, comprising the steps of: Providing a mammary stem cell population; providing a culture medium comprising an ErbB3 / 4 ligand, one or more FGFR2b ligands, and a Wnt agonist; contacting the stem cells with a culture medium; and Culturing the cells under appropriate conditions. [The present invention 1002] The method of any one of claims 10 to 12, wherein the ErbB3 / 4 ligand is a neuregulin polypeptide. [The present invention 1003] A culture medium comprising an ErbB3 / 4 ligand, and one or more FGFR2b ligands, and a Wnt agonist. [The present invention 1004] A culture medium comprising an ErbB3 / 4 ligand suitable for expanding a population of mammary epithelial stem cells for at least four passages. [The present invention 1005] The culture medium of the present invention 1004, further comprising one or more FGFR2b ligands and Wnt agonists. [The present invention 1006] The culture medium of any of claims 1003 to 1005, wherein the ErbB3 / 4 ligand is a neuregulin polypeptide. [The present invention 1007] The culture medium of the present invention 1006, wherein the neuregulin polypeptide is encoded by NRG1. [The present invention 1008] The culture medium of invention 1006 or invention 1007, wherein the neuregulin polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:27. [The present invention 1009] The culture medium of any of claims 1003 and 1005 to 1008, wherein the one or more FGFR2b ligands are FGF7 and / or FGF10. [The present invention 1010] The culture medium of any one of claims 1003 and 1005 to 1009, wherein the Wnt agonist is an Lgr5 agonist. [The present invention 1011] The culture medium of the present invention 1010, wherein the Lgr5 agonist is an Rspondin or an Rspondin mimetic. [The present invention 1012] The culture medium of any one of claims 1003 to 1011, further comprising a BMP inhibitor. [The present invention 1013] The culture medium of any one of claims 1003 to 1012, further comprising one or more components selected from the group consisting of B27, N-acetylcysteine, nicotinamide, a ROCK inhibitor, a TGF-β inhibitor, and a p38 inhibitor. [The present invention 1014] The culture medium of any of claims 1003 to 1012, further comprising B27, N-acetylcysteine, nicotinamide, a ROCK inhibitor, a TGF-β inhibitor, and a p38 inhibitor. [The present invention 1015] (a) the BMP inhibitor is Noggin, and / or (b) the ROCK inhibitor is Y-27632, and / or (c) the TGF-β inhibitor is A83-01, and / or (d) the p38 inhibitor is SB202190; The culture medium of any one of 1012 to 1014 of the present invention. [The present invention 1016] The culture medium of any of claims 1003 to 1015, further comprising one or more components selected from the group consisting of one or more additional receptor tyrosine kinase ligands and p53 stabilizers. [The present invention 1017] (a) the one or more further receptor tyrosine kinase ligands are EGF, PDGF, and / or amphiregulin, and / or (b) the p53 stabilizer is a member of the Nutlin family; The culture medium of the present invention 1015 or 1016. [The present invention 1018] (a) the PDGF is PDGF-CC, and / or (b) the Nutlin family member is Nutlin-3; Culture medium of the present invention 1017. [The present invention 1019] The method of the present invention 1001 or 1002, wherein the culture medium is any one of the culture media of the present inventions 1003 to 1018. [The present invention 1020] Breast organoids containing a population of mammary epithelial stem cells. [The present invention 1021] The breast organoid of the present invention 1020, obtainable or obtained by the method of any of the present invention 1001, 1002, or 1019. [The present invention 1022] The breast organoid of the present invention 1020 or 1021 obtained from normal breast tissue, primary breast cancer, or metastatic breast cancer, which has been passaged for at least four passages. [The present invention 1023] (a) a cyst-like structure, optionally with a central lumen; or (b) A structure resembling a bunch of grapes The breast organoid of any of 1020 to 1022 of the present invention, [The present invention 1024] (a) have continuous epithelial and cell-cell junctions; and / or (b) containing luminal cells but not basal cells; A breast organoid according to any one of claims 1020 to 1023. [The present invention 1025] The breast organoid of any one of 1020 to 1024 of the present invention, which is a breast cancer organoid. [The present invention 1026] Any of the breast organoids of the present invention 1020 to 1024 obtained from normal tissue. [The present invention 1027] A breast organoid according to any one of claims 1020 to 1026, which is in a culture medium according to any one of claims 1003 to 1018. [The present invention 1028] Use of the organoid of any of 1020 to 1026 of the present invention, or cells derived therefrom, in drug discovery screening; toxicity assays; investigation of histogenesis, cell lineages, or differentiation pathways; studying gene expression, including recombinant gene expression; investigating mechanisms involved in tissue damage or repair; investigating inflammatory and infectious diseases; studying mechanisms of pathogenesis; or studying mechanisms of cell transformation or the etiology of cancer. [The present invention 1029] 1027. The organoid of any one of claims 1020 to 1026 or a cell derived from said organoid for use in medicine. [The present invention 1030] Culture medium containing p53 stabilizers. [Brief description of the drawings]

[0018] [Figure 1] (A) Breast tumor tissues obtained from patients are shown according to subtype (estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2) status). (B) Established or promising organoid lines are shown according to subtype (ER, PR, and HER2 status). [Diagram 2] Phase contrast images of established and promising breast (A) tumor organoid lines and (B) normal organoid lines. Scale bars equal 150 μm. [Figure 3a-1] Example of medium optimization. Normal and tumor breast cells from patient W855 were suspended in Cultrex® Basement Membrane Extract (Trevigen, Inc.) and supplemented with the indicated medium. Phase contrast images are shown at (A) passage 0, day 4 and (B) passage 0, day 14. The most beneficial culture conditions are marked with a thick grey box. Image width: 1.5 mm upper panel, 300 μm lower panel. [Figure 3a-2] See the description of Figure 3a-1. [Figure 3b-1] See the description of Figure 3a-1. [Figure 3b-2]See the description of Figure 3a-1. [Figure 4] Aneuploid karyotypes of established breast tumor organoid lines. [Diagram 5] Histological and immunohistochemical comparison of original mammary tissues (W854 tumor (W854T), W855 normal and tumor (W855N and W855T), W859 tumor (W859T)) and organoid lines derived from each. Scale bars equal 100 μm (overall view) and 20 μm (detailed view). Abbreviations: HE=Hematoxylin-eosin staining; PR=Progesterone receptor (clone PgR 636, M356929 Dako), ER=Estrogen receptor alpha (clone SP1, abl6660 Abcam), HER2=Human epidermal growth factor receptor 2 (c-erbB2) (clone SP3, #RM-9103, Thermoscientific). [Figure 6] Immunofluorescence analysis of whole-mount normal (W855) and tumor (W854, W855, and W859) breast organoid lines. Nuclear counterstain is DRAQ5 (DR50050 Biostatus limited). Scale bar equals 100 μm. Abbreviations: Krt8 = keratin-8 (clone Ks8.7, sc101459 Santa Cruz), Krt14 = keratin-14 (clone AF64, PRB-155P Covance), ER = estrogen receptor alpha (clone C1355, Fisher 50-172-167), E-cadherin (clone 36 / E-cadherin, 61082 BD Biosciences), HER2 = human epidermal growth factor receptor 2 (c-erbB2) (clone SP3, #RM-9103, Thermoscientific), PR = progesterone receptor (clone PgR 636, M356929 Dako). [Figure 7] Quantitative PCR on organoids for basal (KRT5 / 14), luminal (KRT8), epithelial (CDH1), and mesenchymal (VIM, ZEB1) markers normalized to the normal breast organoid line W855N. [Figure 8-1]Phase contrast images of breast tumor organoid lines W854T, W855T, and W859T during an experimental drug screen with increasing doses of the EGFR inhibitor Iressa or the p53 stabilizer Nutlin-3. Visibly affected organoids are marked with thick black boxes. Images are 500 μm wide. [Figure 8-2] See description of Figure 8-1. [Figure 8-3] See description of Figure 8-1. [Figure 9-1] Viability of breast tumor organoid lines W854T, W855T, and W859T treated with increasing doses of the EGFR inhibitor Iressa or the p53 stabilizer Nutlin-3 for 7 days as measured by a luminescent ATP assay (CellTiter-Glo 2.0, G9241 Promega). [Figure 9-2] See description of Figure 9-1. [Figure 10] Normal human breast organoids in culture medium as described in Pasic et al. compared to organoids derived from the same patient grown in basal F7 / 10E medium. [Figure 11] Mouse mammary tumor organoids. Phase contrast images of organoids established from KB2P mammary tumors lacking Brca2 and p53, passage 4. Image widths are 7 mm, 1.5 mm, and 600 μm. [Figure 12] Human metastatic breast tumor organoids. Phase contrast images of organoids established from human breast cancer metastasis to the skin, passage 5. Image widths are 7 mm, 1.5 mm, and 600 μm. [Figure 13-1] Overview of mammary organoid culture. [Figure 13-2] See description of Figure 13-1. [Figure 14] Organoid culture medium. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Detailed Description Methods for culturing epithelial stem cells derived from various tissues have been previously described in WO2010 / 090513, WO2012 / 014076, and WO2012 / 168930. The inventors have surprisingly discovered that the addition of ErbB3 / 4 ligand to the culture medium allows mammary epithelial stem cells to be cultured for an increased number of passages compared to when ErbB3 / 4 ligand is absent from the medium.

[0020] Advantageously, the cells and resulting organoids have the ability to survive for long periods of time and to increase in passage number, making it possible to obtain many more cells from a collection of starting cells than was possible using previous methods. This allows for a large number of cells to be obtained for a variety of applications, for example, for drug screening, where a large amount of material is required to test a variety of different drugs. For such applications, where it is necessary to compare results between experiments, the ability to generate the cells from one starting source is advantageous. Furthermore, the improved ability to obtain many more cells from a small collection of starting cells (e.g., a collection of about 100-300 cells) is advantageous for applications where little starting material is available, for example, biopsies of primary or metastatic cancers or circulating tumor cells. In turn, this means that many cells are available for use in transplantation, for example, in breast reconstruction or breast enhancement, and that cells obtained from one useful donor may be transplanted into multiple patients.

[0021] When said cells are cultured in the medium of the present invention, said cells can be multiplied while maintaining stem cell or progenitor cell phenotype.This is referred to as proliferation herein.The organoids containing these stem cells or progenitor cells are formed.Therefore, the use of the medium of the present invention is advantageous for preparing an increased number of these useful stem cells or progenitor cells and for obtaining the organoids containing these cells.

[0022] Thus, a method is provided for culturing mammary epithelial stem cells, comprising culturing one or more mammary epithelial stem cells in contact with an extracellular matrix in the presence of a culture medium comprising a basal medium for animal cells or human cells supplemented with one or more ErbB3 / 4 ligands.

[0023] The culture medium used in the methods of the present invention is preferably a culture medium of the present invention as described herein.

[0024] Thus, for example, there is provided a culture medium comprising a basal medium for animal or human cells to which is added one or more (e.g., one, two, three, four or more) ErbB3 / 4 ligands and one or more (e.g., one, two, three, four or more) FGFR2b ligands for use in the above methods, preferably wherein the one or more FGFR2b ligands are FGF7 and / or FGF10.

[0025] Thus, for example, culture media comprising basal medium for animal or human cells supplemented with one or more (e.g., one, two, three, four or more) ErbB3 / 4 ligands and one or more (e.g., one, two, three, four or more) Wnt agonists for use in the above methods are also provided.

[0026] In some embodiments, the one or more Wnt agonists are Lgr5 agonists, Lgr4 agonists, Lgr6 agonists, or Wnt3a.In some embodiments, the Lgr5 agonist is Rspondin, for example, any one of Rspondin1-4.

[0027] In a preferred embodiment, the culture medium of the present invention comprises a basal medium for animal or human cells, to which one or more (e.g., one, two, three, four or more) ErbB3 / 4 ligands, one or more (e.g., one, two, three, four or more) FGFR2b ligands and one or more (e.g., one, two, three, four or more) Wnt agonists are added, preferably, one or more FGFR2b ligands are FGF7 and / or FGF10, preferably, one or more Wnt agonists are Lgr5 agonists, preferably, Lgr5 agonists are Rspondin, for example, any one of Rspondin1-4.

[0028] In some embodiments, the culture medium of the present invention or the culture medium used in the method of the present invention comprises one or more additional components selected from FGFR2b ligand (e.g., FGF7 and / or FGF10), Lgr5 agonist (e.g., Rspondin, e.g., any one of Rspondin1-4), BMP inhibitor (e.g., Noggin), B27, N-acetylcysteine, nicotinamide, ROCK inhibitor, TGF-β inhibitor (e.g., ALK4, 5, 7 inhibitor, e.g., A83-01), and p38 MAP kinase inhibitor (e.g., SB202190).In some embodiments, the culture medium further comprises at least two, at least three, at least four, at least five, at least six, at least seven (e.g., two, three, four, five, six, seven) of these additional components.In some embodiments, the culture medium further comprises all of these additional components.

[0029] In some embodiments, the culture medium further comprises an additional receptor tyrosine kinase ligand, for example, EGF.

[0030] In some embodiments, the culture medium further comprises a Wnt agonist, for example, Wnt3a.

[0031] In some embodiments, the culture medium further comprises one or more of amphiregulin, TGF-α, and PDGF-CC.For example, in some embodiments, the culture medium further comprises PDGF-CC.In some embodiments, the culture medium further comprises amphiregulin, TGF-α, and PDGF-CC.Therefore, the present invention provides the use of ErbB3 / 4 ligand for culturing breast epithelial stem cells.

[0032] The present invention also provides methods for culturing mammary epithelial stem cells using a growth medium described in WO2012 / 168930, WO2010 / 09013, or WO2012 / 014076 supplemented with at least one (e.g., one, two, three, four, or more than four) ErbB3 / 4 ligand and at least one (e.g., one, two, three, four, or more than four) FGFR2b ligand.

[0033] The inventors have also surprisingly found that adding a p53 stabilizer to the culture medium can ensure that the cell population is predominantly tumor cells. Without wishing to be bound by any theory, it is believed that the p53 stabilizer increases the cellular concentration of p53 (e.g., by blocking the interaction between p53 and Mdm2), stimulates p53-dependent expression, and induces cellular senescence. p53 mutations usually occur in tumor cells. Mutations in p53 can result in mutant p53 proteins that cannot be stabilized by p53 stabilizers. Thus, it is believed that a subset of tumor cells with mutant p53 proteins escapes p53 stabilizer-induced senescence and grows faster than normal cells in a mixed population.

[0034] Thus, the present invention further provides the use of a p53 stabilizing agent for culturing tumor cells.

[0035] The present invention also provides a method for culturing tumor stem cells, using a growth medium as described herein or as described in WO2012 / 168930, WO2010 / 090513, or WO2012 / 014076, to which at least one (e.g., one, two, three, four, or more than four) p53 stabilizer is added. The present invention also provides a culture medium comprising a p53 stabilizer. Preferably, the culture medium is a culture medium as described herein or as described in WO2012 / 168930, WO2010 / 090513, or WO2012 / 014076. Preferably, the culture medium is suitable for culturing epithelial stem cells.

[0036] Preferred p53 stabilizer is a member of Nutlin family, for example Nutlin-1, Nutlin-2 or Nutlin-3.For example, in some embodiments, p53 stabilizer is Nutlin-3.Other p53 stabilizers (for example CP-31398) are known in the art.Therefore, those skilled in the art can use any of these p53 stabilizers.

[0037] ErbB3 / 4 Ligand The ErbB receptor tyrosine kinase family consists of four cell surface receptors: i) ErbB1 / EGFR / HER1, ii) ErbB2 / HER2, iii) ErbB3 / HER3, and iv) ErbB4 / HER4. An ErbB3 / 4 ligand is defined herein as a ligand that can bind to ErbB3 and / or ErbB4. Thus, EGF is not an ErbB3 / 4 ligand because it does not bind to ErbB3 / 4. In some embodiments, an ErbB3 / 4 ligand binds to ErbB3 and does not bind to ErbB4. In some embodiments, an ErbB3 / 4 ligand binds to ErbB4 and does not bind to ErbB3. In some embodiments, the binding of an ErbB3 / 4 ligand to ErbB3 or ErbB4 induces heterodimerization of said ErbB3 or said ErbB4 with ErbB2. In some embodiments, induction of heterodimerization of ErbB3 or ErbB4 with ErbB2 stimulates intrinsic kinase activity and phosphorylates tyrosines. In the context of the culture medium of the present invention, the ErbB3 / 4 ligand is known as "N".

[0038] A variety of ErbB3 / 4 ligands are known in the art. In a preferred embodiment, one or more ErbB3 / 4 ligands of culture medium are members of the neuregulin / heregulin family. The neuregulin / heregulin family is referred to herein as the neuregulin family. The neuregulin family is a family of structurally related polypeptide growth factors that are gene products of alternatively spliced ​​genes NRG1, NRG2, NRG3, and NRG4. In a further preferred embodiment, one or more ErbB3 / 4 ligands of culture medium are polypeptides that are gene products of one or more of NRG1, NRG2, NRG3, and NRG4 (i.e., neuregulin polypeptides). In some embodiments, ErbB3 / 4 ligands are polypeptides that are gene products of NRG1. The polypeptides that are the gene products of NRG1, NRG2, NRG3, and NRG4 can be any isoform resulting from alternative splicing of NRG1 mRNA, NRG2 mRNA, NRG3 mRNA, or NRG4 mRNA. Thus, for example, the polypeptides that are the gene products of NRG1 can be any of the following isoforms: NRG1 type I (also known as heregulin, NEU differentiation factor (NDF), or acetylcholine receptor inducing activity (ARIA)), NRG1 type II (also known as glial growth factor-2 (GGF2)), NRG1 type III (also known as Sensory and motor neuron-derived factor (SMDF)), NRG1 type IV, NRG1 type V, or NRG1 type VI. In some embodiments, the neuregulin polypeptide is proneuregulin-1, membrane-bound isoform isoform HRG-β1 (NP_039250.2), proneuregulin-1, membrane-bound isoform isoform HRG-β1b (NP_001153471.1), proneuregulin-1, membrane-bound isoform isoform HRG-β1c (NP_001153467.1), proneuregulin-1, or membrane-bound isoform isoform HRG-β1d (NP_001153473.1).

[0039] In some embodiments, the ErbB3 / 4 ligand is derived from a human. Thus, in some embodiments, the ErbB3 / 4 ligand is a human gene product of one or more of NRG1, NRG2, NRG3, and NRG4 (i.e., a human neuregulin polypeptide).

[0040] In some embodiments, the NRG1 gene has the sequence of Gene ID: 3084 or NG_012005.1. In some embodiments, the NRG2 gene has the sequence of Gene ID: 9542. In some embodiments, the NRG3 gene has the sequence of Gene ID: 10718 or NG_013373.1. In some embodiments, the NRG4 gene has the sequence shown in Gene ID: 145957.

[0041] In some embodiments, the neuregulin polypeptide is the gene product of NRG1, and is selected from the group consisting of NP_001153467.1 (SEQ ID NO:1), NP_001153468.1 (SEQ ID NO:2), NP_001153471.1 (SEQ ID NO:3), NP_001153473.1 (SEQ ID NO:4), NP_001153474.1 (SEQ ID NO:5), NP_001153476.1 (SEQ ID NO:6), NP_001153477.1 (SEQ ID NO:7), NP_001153479.1 (SEQ ID NO:8), NP_001153480.1 (SEQ ID NO:9), NP_004486.2 (SEQ ID NO:10), NP_004487.1 (SEQ ID NO:11), NP_001153489.1 (SEQ ID NO:12), NP_001153490.1 (SEQ ID NO:13), NP_001153491.1 (SEQ ID NO:14), NP_001153492.1 (SEQ ID NO:15), NP_001153493.1 (SEQ ID NO:16), NP_001153494.1 (SEQ ID NO:17), NP_001153495.1 (SEQ ID NO:18), NP_001153496.1 (SEQ ID NO:19), NP_001153497.1 (SEQ ID NO:20), NP_001153498.1 (SEQ ID NO:21), NP_001153499.1 (S NO:10), NP_039250.2 (SEQ ID NO:11), NP_039251.2 (SEQ ID NO:12), NP_039252.2 (SEQ ID NO:13), NP_039253.1 (SEQ ID NO:14), NP_039254.1 (SEQ ID NO:15), NP_039256.2 (SEQ ID NO:16), or NP_039258.1 (SEQ ID NO:17). In some embodiments, the neuregulin polypeptide is the gene product of NRG2 and has a sequence set forth in NP_001171864.1 (SEQ ID NO:18), NP_004874.1 (SEQ ID NO:19), NP_053584.1 (SEQ ID NO:20), NP_053585.1 (SEQ ID NO:21), or NP_053586.1 (SEQ ID NO:22). In some embodiments, the neuregulin polypeptide is the gene product of NRG3 and has a sequence set forth in NP_001010848.2 (SEQ ID NO:23), NP_001159444.1 (SEQ ID NO:24), or NP_001159445.1 (SEQ ID NO:25). In some embodiments, the neuregulin polypeptide is the gene product of NRG4 and has the sequence set forth in NP_612640.1 (SEQ ID NO:26).

[0042] In some embodiments, the at least one ErbB3 / 4 ligand is a biologically active variant of one or more naturally occurring ErbB3 / 4 ligands, such as one or more neuregulin family members. Neuregulin variants can be naturally occurring (e.g., allelic variants occurring at the NRG1 locus) or recombinantly produced, and have the same, similar, or substantially similar amino acid sequence as a neuregulin polypeptide.

[0043] In some embodiments, a neuregulin variant has a sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a neuregulin polypeptide. For example, a neuregulin variant has a sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs:1-26.

[0044] In some embodiments, the at least one ErbB3 / 4 ligand is at least one biologically active fragment of at least one naturally occurring ErbB3 / 4 ligand, e.g., a biologically active fragment of one or more neuregulin polypeptides.

[0045] "Biologically active" is defined herein to mean that an ErbB3 / 4 ligand, e.g., a variant or fragment, is capable of binding to ErbB3 and / or ErbB4, optionally, upon binding to ErbB3 and / or ErbB4, heterodimerization of said ErbB3 or said ErbB4 with ErbB2 is induced, and optionally, upon induction of heterodimerization of ErbB3 or ErbB4 with ErbB2, stimulates intrinsic kinase activity resulting in tyrosine phosphorylation.

[0046] In some embodiments, the ErbB3 / 4 ligand is a fragment of a polypeptide having an amino acid sequence set forth in any one of SEQ ID NOs:1-26, wherein the fragment comprises at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 amino acids of the sequence set forth in any one of SEQ ID NOs:1-26.

[0047] In some embodiments, the ErbB3 / 4 ligand is a variant of a polypeptide having an amino acid sequence set forth in any one of SEQ ID NOs: 1-26, said variant having an amino acid sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a polypeptide having an amino acid sequence set forth in any one of SEQ ID NOs: 1-26.

[0048] References to percent sequence identity between two amino acid sequences mean that when the two sequences are aligned, the percentage of amino acids that are the same when comparing the two sequences. This alignment and percent homology or sequence identity can be determined using software programs known in the art, such as those described in Section 7.7.18 of Current Protocols in Molecular Biology (FM Ausubel et al, eds., 1987) Supplement 30. Preferred alignments are ascertained by the Smith-Waterman homology search algorithm, using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2, with a BLOSUM matrix of 62. The Smith-Waterman homology search algorithm is disclosed in Smith & Waterman (1981) Adv. Appl. Math. 2: 482-489.

[0049] In some embodiments, the ErbB3 / 4 ligand is a variant of a fragment of a polypeptide having an amino acid sequence as set forth in any one of SEQ ID NOs: 1-26, said fragment comprising at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 amino acids of the sequence as set forth in any one of SEQ ID NOs: 1-26, said variant having an amino acid sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to said fragment.

[0050] In some embodiments, the ErbB3 / 4 ligand is a fragment of a biologically active variant of a polypeptide having an amino acid sequence set forth in any one of SEQ ID NOs: 1-26, said variant having an amino acid sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a polypeptide having an amino acid sequence set forth in any one of SEQ ID NOs: 1-26, said fragment comprising at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 amino acids of said variant.

[0051] In some embodiments, ErbB3 / 4 ligand is non-natural ligand, for example, synthetic ligand or anti-ErbB3 / 4 antibody.Methods for generating antibodies against target of interest are well known in the art.In some embodiments, anti-ErbB3 / 4 antibody is agonistic anti-ErbB3 / 4 antibody.Preferably, antibody is biologically active.Any suitable antibody can be used, for example, as described herein.

[0052] ErbB3 / 4 ligands can be identified by methods known in the art, such as by binding GST-tagged candidate ligands to ErbB receptors expressed in insect cells and phosphorylating their tyrosines (Carraway 3 rd et al., (1997) Nature 387(6632):512-6).

[0053] In some embodiments, ErbB3 / 4 ligand comprises EGF domain or EGF-like domain. EGF and EGF-like domain are evolutionarily conserved protein domains recognized in the art (see, for example, Wouters et al. (2005) Protein Sci. 14(4): 1091-1103). Thus, in some embodiments where ErbB3 / 4 ligand is the biologically active fragment of at least one native ErbB3 / 4 ligand, the fragment comprises EGF domain or EGF-like domain.

[0054] In some embodiments, the ErbB3 / 4 ligand is human neuregulin beta-1. In some embodiments, the human neuregulin beta-1 has the amino acid sequence shown in SEQ ID NO:27. Human neuregulin beta-1 is a fragment of a neuregulin polypeptide, the fragment including an EGF-like domain. In preferred embodiments, the ErbB3 / 4 ligand includes the EGF-like domain of human neuregulin beta-1 or consists of the EGF-like domain of human neuregulin beta-1.

[0055] In some embodiments, the ErbB3 / 4 ligand has biological activity similar to recombinant human neuregulin beta-1.

[0056] In some embodiments, the ErbB3 / 4 ligand is a fragment of a polypeptide having the amino acid sequence set forth in SEQ ID NO:27, the fragment comprising at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 62, or at least 64 amino acids of the sequence set forth in SEQ ID NO:27.

[0057] In some embodiments, the ErbB3 / 4 ligand is a variant of a polypeptide having an amino acid sequence set forth in SEQ ID NO:27, which variant has an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a polypeptide having an amino acid sequence set forth in SEQ ID NO:27.

[0058] In some embodiments, the ErbB3 / 4 ligand is a variant of a fragment of a polypeptide having the amino acid sequence set forth in SEQ ID NO:27, said fragment comprising at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 62, or at least 64 amino acids of the sequence set forth in SEQ ID NO:27, said variant having an amino acid sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to said fragment.

[0059] In some embodiments, the ErbB3 / 4 ligand is a fragment of a biologically active variant of a polypeptide having an amino acid sequence as set forth in SEQ ID NO:27, said variant having an amino acid sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a polypeptide having an amino acid sequence as set forth in SEQ ID NO:27, said fragment comprising at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 62, or at least 64 amino acids of said variant.

[0060] The ErbB3 / 4 ligand may be present at any suitable concentration. For example, the ErbB3 / 4 ligand may be present at a concentration of 0.05-500nM, 0.05-100nM, 0.05-50nM, 0.05-10nM, 0.05-5nM, 0.05-1nM, 0.5-500nM, 0.5-100nM, 0.5-50nM, 0.5-10nM, 0.5nM-1nM, 1-10nM, 3-10nM, 3-8nM, or 4-6nM. For example, the ErbB3 / 4 ligand may be present at a concentration of about 5nM. In some embodiments, the ErbB3 / 4 ligand is used at a concentration of at least 0.05 nM, at least 0.5 nM, at least 1 nM, at least 3 nM, at least 4 nM, at least 5 nM, at least 10 nM, at least 50 nM, or at least 100 nM.

[0061] In addition to the ErbB3 / 4 ligand, cell culture media generally contain a number of components necessary to support the maintenance and / or growth of cultured cells. Thus, the cell culture media of the present invention generally contain many other components in addition to the ErbB3 / 4 ligand. The appropriate combination of components can be easily formulated by those skilled in the art in light of the disclosure herein. The culture medium according to the present invention is generally a nutrient solution containing standard cell culture components, such as amino acids, vitamins, inorganic salts, carbon energy sources, and buffers, as described in more detail below. Other standard cell culture components that may be included in the culture include hormones, such as progesterone, proteins, such as albumin, catalase, insulin, and transferrin. These other standard cell culture components constitute the "basal" culture medium.

[0062] The culture medium according to the present invention may be produced by improving an existing cell culture medium. Those skilled in the art will understand from general knowledge the type of culture medium that may be suitable for improving for use in epithelial stem cell culture. Suitable cell culture media are commercially available and include, but are not limited to, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Knockout-DMEM (KO-DMEM), Glasgow Minimum Essential Medium (G-MEM), Basal Medium Eagle (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, Medium 199, and RPMI1640 medium. Examples of suitable "basal" cultures are also given in WO2012 / 168930, WO2010 / 090513, and WO2012 / 014076.

[0063] In some embodiments, the basal medium is AdDF+++. In some embodiments, AdDF+++ includes Advanced DMEM / F12 (Dulbecco's Modified Eagle's Medium / Ham's F-12), GlutaMax, HEPES, penicillin / streptomycin, primocin.

[0064] In some embodiments, the base medium is D10F. In some embodiments, D10F comprises 31966-DMEM (Dulbecco's Modified Eagle's Medium), fetal bovine serum (FBS), and penicillin / streptomycin.

[0065] Therefore, in some embodiments, one of these existing cell culture media is used as the basic culture medium to which one or more ErbB3 / 4 ligands are added.As mentioned above, the culture medium of the present invention further comprises one or more ligands of FGFR2b (for example, FGF7 and / or FGF10) and / or one or more Wnt agonists (preferably agonistic Lgr5 ligand, preferably Rspondin).In some embodiments, the culture medium of the present invention comprises the following additional components: (i) FGF7 and / or FGF10 and (ii) Rspondin.In some embodiments, the culture medium of the present invention comprises the following additional components: (i) FGF7 and / or FGF10, (ii) Rspondin and (iii) one or more additional receptor tyrosine kinase ligands.

[0066] In some embodiments, the culture medium of the present invention further comprises one or more components selected from a BMP inhibitor (e.g., Noggin), B27, N-acetylcysteine, nicotinamide, a ROCK inhibitor, a TGF-β inhibitor (e.g., A83-01), a p38 MAP kinase inhibitor (e.g., SB202190).

[0067] In some embodiments, the culture medium of the present invention comprises ErbB3 / 4 ligand, Rspondin, BMP inhibitor (e.g., Noggin), B27, N-acetylcysteine, nicotinamide, ROCK inhibitor, TGF-β inhibitor (e.g., A83-01), p38 MAP kinase inhibitor (e.g., SB202190), FGF7, and FGF10, and optionally comprises one or more additional components selected from one or more additional receptor tyrosine kinase ligands (e.g., EGF, amphiregulin, TGF-α, PDGF), p53 stabilizer, and Wnt agonist (e.g., Wnt3a).

[0068] As will be evident to the skilled reader, the preferred culture method of the present invention is advantageous since no feeder cells are required. Feeder cell layers are often used to support the culture of stem cells and inhibit the differentiation of stem cells. A feeder cell layer is generally a monolayer of cells that is co-cultured with the cells of interest and provides a suitable surface for growing the cells of interest. The feeder cell layer provides an environment in which the cells of interest can grow. The feeder cells are often mitotically inactivated (e.g., by irradiation or mitomycin C treatment) to prevent their proliferation. The use of feeder cells is undesirable since it complicates the passaging of cells (cells must be separated from the feeder cells for each passaging and new feeder cells are required for each passaging). The use of feeder cells can also lead to contamination of the desired cells with the feeder cells. This is clearly problematic for any medical application and complicates the analysis of the results of any experiments performed on the cells, even in an investigative situation. The culture medium of the present invention is particularly advantageous since it can be used to culture cells without contact with feeder cells. That is, the methods of the present invention do not require a feeder cell layer to support the cells whose growth is being funded.

[0069] Therefore, the composition of the present invention may be a feeder cell-free composition.Conventionally, a composition is considered to be feeder cell-free if the cells in the composition have been cultured for at least one passage in the absence of a feeder cell layer.The feeder cell-free composition of the present invention usually contains less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1% feeder cells (expressed as a percentage of the total number of cells in the composition), or preferably does not contain any feeder cells at all.

[0070] BMP inhibitors The culture medium of the present invention may contain one or more bone morphogenetic protein (BMP) inhibitors.

[0071] In some embodiments, the culture medium may contain any suitable BMP inhibitor. BMP binds as a dimeric ligand to a receptor complex consisting of two different receptor serine / threonine kinases, type I receptor and type II receptor. Type II receptor phosphorylates type I receptor, which results in the activation of this receptor kinase. Type I receptor then phosphorylates specific receptor substrates (SMADs), resulting in signal transduction pathways that lead to transcriptional activation. Advantageously, BMP inhibitors promote the expression of Lgr5. Therefore, the presence of BMP inhibitors in the culture medium of the present invention is more likely to result in more proliferative organoids than in the absence of BMP inhibitors. In some embodiments, the Lgr5 expression of cells cultured with BMP inhibitors is upregulated compared to cells not cultured with BMP inhibitors. Thus, the addition of BMP inhibitors typically results in more proliferative organoids.

[0072] BMP inhibitors are defined as agents that bind to BMP molecules to form complexes. In this case, for example, the binding of BMP molecules to BMP receptors is blocked or inhibited, thereby neutralizing BMP activity. Alternatively, the inhibitors are agents that act as antagonists or reverse agonists. This type of inhibitor binds to BMP receptors and blocks the binding of BMP to the receptors. An example of the latter agent is an antibody that binds to BMP receptors and blocks the binding of BMP to the receptor that binds to the antibody.

[0073] BMP inhibitor may be added to culture medium in an amount effective to inhibit BMP-dependent activity in cells up to 90%, more preferably up to 80%, more preferably up to 70%, more preferably up to 50%, more preferably up to 30%, more preferably up to 10%, more preferably up to 0% when compared with the BMP activity level in the absence of said inhibitor when evaluated in the same cell type.As known to those skilled in the art, BMP activity can be confirmed by measuring the transcription activity of BMP, for example, as exemplified in Zilberberg et al., 2007.BMC Cell Biol. 8:41.

[0074] Several natural BMP binding protein classes are known, including Noggin (Peprotech), chordin and chordin-like proteins containing a chordin domain (R&D systems), follistatin and follistatin-related proteins containing a follistatin domain (R&D systems), DAN and DAN-like proteins containing a DAN cysteine-knot domain (R&D systems), sclerostin / SOST (R&D systems), decorin (R&D systems), and alpha-2 macroglobulin (R&D systems).

[0075] Examples of BMP inhibitors for use in the method of the present invention are Noggin, DAN, and DAN-like proteins, including Cerberus and Gremlin (R&D systems).These diffusible proteins can bind to BMP ligands with varying degrees of affinity, and inhibit BMP ligands from approaching signal transduction receptors.Adding any of these BMP inhibitors to basic culture medium prevents stem cell loss.

[0076] A preferred BMP inhibitor is Noggin. Thus, in some embodiments, the culture medium described herein comprises Noggin. In the context of the culture medium of the present invention, Noggin is also referred to herein as "No". Noggin is preferably added to the base culture medium at a concentration of at least 10ng / ml, for example at least 20ng / ml, more preferably at least 25ng / ml. Even more preferably, the concentration is about 25ng / ml.

[0077] During the cultivation of stem cells, said BMP inhibitor can be added to culture medium when necessary, for example, every day or every other day.BMP inhibitor is preferably added to culture medium every other day.Culture medium can be renewed when necessary, for example, every day or every other day.

[0078] Wnt agonists The culture medium of the present invention may contain one or more Wnt agonists. The Wnt signaling pathway is defined by a series of events that occur when the cell surface Wnt receptor complex, which includes Frizzled receptor, LRP, and LGR, is activated, usually by an extracellular signaling molecule, such as a Wnt family member. This activates the Dishevelled family proteins, which inhibit a protein complex that includes axin, GSK-3, and the protein APC, which degrades intracellular β-catenin. The resulting high concentration of nuclear β-catenin enhances transcription by TCF / LEF family transcription factors.

[0079] Wnt agonist is defined as a drug that activates TCF / LEF-mediated transcription in cells.Therefore, Wnt agonist is selected from true Wnt agonist that binds to and activates Wnt receptor complex, including any and all of Wnt family proteins, intracellular β-catenin degradation inhibitors, GSK inhibitors (e.g., CHIR9901), and TCF / LEF activators.

[0080] 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, WnM1, and Wnt-16. A summary of human Wnt proteins is shown 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 negatively regulate the level of Wnt receptor complexes at the membrane, possibly by ubiquitination of Frizzled. Therefore, the inventors hypothesize that inhibition of RNF43 or ZNRF3 by antagonistic antibodies, RNAi, or small molecule inhibitors indirectly stimulates the Wnt pathway. RNF43 and ZNRF3 have catalytic cyclic domains (with ubiquitination activity). This catalytic cyclic domain can be targeted in small molecule inhibitor design. Several anti-RNF43 antibodies and several anti-ZNRF3 antibodies are commercially available. In some embodiments, such antibodies are suitable Wnt agonists in the context of the present invention.

[0081] In a preferred embodiment, the Wnt agonist in the culture medium is any agonist that can stimulate the Wnt pathway through Lgr5 cell surface receptor.That is, in a preferred embodiment, the Wnt agonist in the culture medium is Lgr5 agonist.Known Lgr5 agonists include Rspondin, its fragments and derivatives, and anti-Lgr5 antibodies (see, for example, WO2012 / 140274 and De Lau, W. et al. Nature, 2011 Jul 4;476(7360):293-7).Preferred Lgr5 agonist is Rspondin.Any suitable Rspondin can be used.For example, Rspondin can be selected from one or more of Rspondin1, Rspondin2, Rspondin3, and Rspondin4, or its derivatives. For example, any of Rspondin1 (NU206, Nuvelo, San Carlos, CA), Rspondin2 ((R&D systems), Rspondin3, and Rspondin4 can be used. Rspondin1, 2, 3, and 4 are also referred to herein as "Rspondin1-4". Rspondin fragments may be used as Wnt agonists. For example, in some embodiments, the Wnt agonist is an Rspondin fragment that comprises or consists of a furin domain. Examples of suitable Rspondin fragments include those having the amino acid sequence set forth in SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, or SEQ ID NO:31, or those having the amino acid sequence set forth in SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, or SEQ ID NO:31. The Lgr5 agonist is represented by a sequence having 70%, 80%, 90%, or more than 99% identity with any one of SEQ ID NO:31. In some embodiments, the Lgr5 agonist is an anti-Lgr5 antibody, more preferably an agonist anti-Lgr5 antibody. An example of an agonist anti-Lgr5 antibody is 1D9 (available from BD Biosciences, BDB562733, No.:562733). Thus, in one embodiment, the agonist is antibody 1D9. The VL of antibody 1D9 is represented by SEQ ID NO:32, and the VH is represented by SEQ ID NO:33.Thus, in one embodiment, the agonist is an antibody comprising or consisting of SEQ ID NO:32 and / or SEQ ID NO:33.

[0082] In some embodiments, the Wnt agonist in culture medium is any agonist that can stimulate the Wnt pathway through Lgr4 cell surface receptor.That is, in some embodiments, the Wnt agonist in culture medium is Lgr4 agonist.

[0083] In some embodiments, the Wnt agonist in culture medium is any agonist that can stimulate the Wnt pathway through Lgr6 cell surface receptor.That is, in some embodiments, the Wnt agonist in culture medium is Lgr6 agonist.

[0084] Wnt agonist may be added to culture medium in an amount effective to stimulate Wnt activity in cells 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 at least 100% compared to the level of Wnt activity in the absence of said molecule when evaluated in the same cell type.As known to those skilled in the art, Wnt activity can be confirmed by measuring Wnt transcription activity, for example, by pTOPFLASH and pFOPFLASH Tcf luciferase reporter constructs (Korinek et al., 1997. Science 275:1784-1787).

[0085] A soluble Wnt agonist, such as Wnt-3a, may be provided in the form of a Wnt conditioned medium. For example, about 10% to about 30%, for example, about 10 ng / ml to about 10 μg / ml, preferably about 1 μg / ml, of the Wnt conditioned medium may be used.

[0086] Rspondins 1 to 4 may be provided in the form of an Rspo conditioned medium. For example, an Rspo conditioned medium having a concentration of about 10% to about 30%, for example, about 10 ng / ml to about 10 μg / ml, preferably about 1 μg / ml, may be used.

[0087] One or more Wnt agonists may be used in the culture medium, for example, two, three, four or more Wnt agonists.In one embodiment, the culture medium comprises an Lgr5 agonist, for example, Rspondin, and further comprises an additional Wnt agonist.In this context, the additional Wnt agonist may be selected, for example, from the group consisting of Wnt-3a, GSK-inhibitor (for example, CHIR99021), Wnt-5, Wnt-6a, and Norrin.In one embodiment, the culture medium comprises Rspondin, and further comprises a soluble Wnt ligand, for example, Wnt3a.The addition of a soluble Wnt ligand has been shown to be particularly advantageous for the proliferation of human epithelial stem cells (as described in WO2012 / 168930).

[0088] Any suitable concentration of Wnt agonist, such as Rspondin, can be used, for example at least 200ng / ml, more preferably at least 300ng / ml, more preferably at least 500ng / ml. Even more preferred Rspondin concentrations are at least 500ng / ml or about 1μg / ml.

[0089] During stem cell culture, the Wnt agonist may be added to the culture medium whenever necessary, for example, every day or every other day. The Wnt agonist is preferably added to the culture medium every other day.

[0090] antibody The antibody used in the present invention, for example, an anti-ErbB3 / 4 antibody, an agonist anti-Lgr5 antibody, or an antagonist TGF-β inhibitor (see below), can be any antibody, fragment, etc. A conventional antibody is composed of two identical heavy chains and two identical light chains linked by disulfide bonds. Each of the heavy and light chains contains a constant region and a variable region. Each of the variable regions contains three CDRs that are mainly responsible for binding to an antigen epitope. These are numbered consecutively from the N-terminus and are called CDR1, CDR2, and CDR3. Of these, the CDR3 region contains the most variable region and usually provides a significant portion of the residues that contact the target. The most highly conserved parts of the variable region are called "framework regions".

[0091] The term antibody is used herein in the broadest sense and covers, in particular, but not limited to, monoclonal antibodies of any isotype, such as IgG, IgM, IgA, IgD, and IgE (including full-length monoclonal antibodies), polyclonal antibodies, including recombinant polyclonal antibodies, oligoclonals, multispecific antibodies, chimeric antibodies, nanobodies, diabodies, BiTEs, Tandabs, mimetobodies, bispecific antibodies, humanized antibodies, human antibodies, deimmunised antibodies, and antibody fragments. Furthermore, the term covers scaffolds, such as anticalins, anchorins, etc. Antibodies that react with specific epitopes of the Lgr protein described above can be made by recombinant methods, such as by selecting recombinant antibody libraries in phage or similar vectors, or by immunizing animals with the Lgr epitopes of nucleic acids encoding the Lgr epitopes.

[0092] In one embodiment, the antibody according to the invention comprises a single domain antibody, F(ab')2, Fab, Fab', or single chain Fv (scFv) fragment. For example, an Fc fragment capable of activating complement and binding to Fc receptors may be present but is not required for antibodies and variants or derivatives thereof. An scFv fragment is an epitope-binding fragment comprising at least one fragment of an antibody heavy chain variable region (VH) linked to at least one fragment of an antibody light chain variable region (VL). The linker may be a short flexible peptide designed to ensure that the correct three-dimensional folding of the VL and VH regions occurs once the VL and VH regions are linked such that the single chain antibody fragment maintains the target molecule binding specificity of the whole antibody. The carboxyl terminus of the VL or VH sequence may be covalently linked to the amino acid terminus of the complementary VL or VH sequence by the linker.

[0093] The antibody may be a diabody, a mimetibody, a nanobody, and / or a bispecific antibody. Nanobodies are single-domain antibodies that occur naturally in camelids. In contrast to standard antibodies, nanobodies are relatively simple proteins that only contain a variable region resembling a heavy chain. Bispecific antibodies are engineered monoclonal antibodies that consist of two separate binding sites and can bind to two different epitopes. Examples of bispecific antibodies are described in more detail below in the sections on dual-targeting agonists and multi-targeting agonists.

[0094] The antibody may be a chimeric antibody that contains the combined heavy and light chain portions, e.g., the variable regions or portions thereof, of a non-human antibody, while the remaining portions, e.g., the heavy and light chain constant regions, are human. Chimeric antibodies can be produced by recombinant processes well known in the art, and have animal variable regions and human constant regions.

[0095] The antibody may be a human antibody or a humanized antibody. The term "human antibody" refers to an antibody whose variable and constant domain sequences are derived from human sequences. In a humanized antibody, only the complementarity determining regions (CDRs) responsible for antigen binding and specificity are derived from an animal and have amino acid sequences corresponding to animal antibodies, while substantially all of the remaining parts of the molecule (except for a small portion of the framework regions, possibly located within the variable regions) are derived from a human and correspond in amino acid sequence to a human antibody. Methods for humanizing non-human antibodies are known in the art. As known to those skilled in the art, an antibody, for example a rat antibody, can be humanized by grafting its CDRs onto the variable light (VL) and variable heavy (VH) frameworks of a human Ig molecule, while retaining the rat framework residues believed to be essential for specificity and affinity. Overall, an antibody with grafted CDRs consists of more than 80% human amino acid sequences.

[0096] In some embodiments, the antibody is a deimmunized antibody in which T cell and B cell epitopes have been removed. Such antibodies are less immunogenic when applied in vivo.

[0097] TGF-β inhibitors The culture medium of the present invention may contain a TGF-β inhibitor. The presence of a TGF-β inhibitor in the medium is advantageous because it enhances the efficiency of human organoid formation. TGF-β signaling is involved in many cell functions, including cell growth, cell fate, and apoptosis. Signaling typically begins with TGF-β superfamily ligand binding to type II receptor, which recruits and phosphorylates type I receptor. Type I receptor then phosphorylates SMAD. SMAD acts as a transcription factor in the nucleus to regulate the expression of target genes.

[0098] TGF-β superfamily ligands include bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs), anti-Mullerian hormone (AMH), activin, nodal, and TGF-β. Generally, Smad2 and Smad3 are phosphorylated by ALK4, 5, and 7 receptors in the TGF-β / activin pathway. In contrast, Smad1, Smad5, and Smad8 are phosphorylated as part of the bone morphogenetic protein (BMP) pathway. Although there may be some crossover between pathways, in the context of the present invention, a "TGF-β inhibitor" or a "TGF-β signaling inhibitor" is preferably an inhibitor of the TGF-β pathway that works through Smad2 and Smad3. Thus, in some embodiments, the TGF-β inhibitor is not a BMP inhibitor, for example, the TGF-β inhibitor is not Noggin. In some embodiments, a BMP inhibitor is added to the culture medium in addition to the TGF-β inhibitor (see above).

[0099] Thus, a TGF-β inhibitor can be any agent that reduces the activity of the TGF-β signaling pathway, preferably the signaling pathway that works through Smad2 and / or Smad3, more preferably the signaling pathway that works through ALK4, ALK5, or ALK7. There are many ways to disrupt the TGF-β signaling pathway that are known in the art and can be used with the present invention. For example, TGF-β signaling may be disrupted by inhibiting TGF-β expression by small interfering RNA strategies; by inhibiting furin (a TGF-β activating protease); by inhibiting the pathway with physiological inhibitors; by neutralizing TGF-β using monoclonal antibodies; by inhibiting TGF-β receptor kinase 1 (also known as activin receptor-like kinase, ALK5), ALK4, ALK6, ALK7, or other TGF-β-related receptor kinases with small molecule inhibitors; by inhibiting Smad2 and Smad3 signaling, for example, by overexpressing Smad7, a physiological inhibitor of these, or by using thioredoxin as a Smad anchor that prevents Smads from being activated (Fuchs, O. Inhibition of TGF-Signaling for the Treatment of Tumor Metastasis and Fibrotic Diseases. Current Signal Transduction Therapy, Volume 6, Number 1, January 2011, pp. 29-43(15)).

[0100] Various methods are known to determine whether a substance is a TGF-β inhibitor and can be used with the present invention. For example, cell assays can be used. In cell assays, cells are stably transfected with reporter constructs that contain human PAI-1 promoter or Smad binding sites to drive luciferase reporter genes. Inhibition of luciferase activity compared to control groups can be used as a measure of compound activity (De Gouville et al., Br J Pharmacol. 2005 May; 145(2): 166-177).

[0101] The TGF-β inhibitor according to the present invention may be a protein, peptide, small molecule, small interfering RNA, antisense oligonucleotide, aptamer, or antibody. The inhibitor may be natural or synthetic. In one embodiment, the TGF-β inhibitor is an inhibitor of ALK4, ALK5, and / or ALK7. For example, the TGF-β inhibitor may bind to ALK4, ALK5, and / or ALK7 and directly inhibit ALK4, ALK5, and / or ALK7. Examples of preferred small molecule TGF-β inhibitors that can be used in the context of the present invention include the small molecule inhibitors listed in Table 1.

[0102] (Table 1) Small molecule TGF-β inhibitors targeting receptor kinases TIFF0007602446000001.tif212146TIFF0007602446000002.tif58146

[0103] In some embodiments, the TGF-β inhibitor is optionally a small molecule inhibitor selected from the group consisting of A83-01, SB-431542, SB-505124, SB-525334, LY364947, SD-208, and SJN2511.

[0104] In some embodiments, more than one TGFβ inhibitor is not present in the medium. In other embodiments, more than one TGFβ inhibitor is present in the medium, for example, two, three, four or more. In some embodiments, the medium of the present invention comprises one or more of any inhibitors listed in Table 1. The medium may comprise any combination of one inhibitor and another inhibitor listed. For example, the medium may comprise SB-525334 or SD-208 or A83-01; or 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 may also inhibit TGF-β receptor kinase at high concentrations. For example, SB-203580 is a p38 MAP kinase inhibitor that is believed to inhibit ALK5 at high concentrations (e.g., about 10 μM or more). Any such inhibitor that inhibits the TGF-β signaling pathway can also be used in the context of the present invention.

[0105] In some embodiments, the TGFβ inhibitor is present at at least 5 nM, e.g., at least 50 nM, at least 100 nM, at least 300 nM, at least 450 nM, at least 475 nM, e.g., between 5 nM and 500 mM, between 10 nM and 100 mM, between 50 nM and 700 uM, between 50 nM and 10 uM, between 100 nM and 1000 nM, between 350 and 650 nM, or more preferably at about 500 nM.

[0106] A83-01 may be added to the medium at a concentration of 10 nM to 10 uM, or 1 uM to 8 uM, or 4 uM to 6 uM. For example, A83-01 may be added to the medium at about 5 uM. Those skilled in the art will know how to determine the concentration of other TGFβ inhibitors for use in the present invention.

[0107] Receptor tyrosine kinase ligands ErbB3 / 4 ligand is a receptor tyrosine kinase ligand. The culture medium of the present invention may further comprise one or more additional receptor tyrosine kinase ligands that are not ErbB3 / 4 ligands themselves. One example of a receptor tyrosine kinase ligand for use in the present invention is EGF, which is the ligand of the receptor tyrosine kinase EGFR. Many receptor tyrosine kinase ligands are also mitogenic growth factors.

[0108] The culture medium of the present invention may contain one or more mitogenic growth factors. The one or more mitogenic growth factors may be selected from the growth factor family including epidermal growth factor (EGF, Peprotech), transforming growth factor-α (TGF-α, Peprotech), basic fibroblast growth factor (bFGF, PEPROTECH), brain-derived neurotrophic factor (BDNF, R&D Systems), platelet-derived growth factor (PDGF, Peprotech), amphiregulin (R&D Systems). A preferred PDGF is PDGF-CC. In the context of the culture medium of the present invention, EGF is also referred to herein as "E" and FGF is also referred to herein as "F".

[0109] In some embodiments, the receptor tyrosine kinase ligand for use in the present invention is one or more mitogenic growth factors selected from human EGF, human PDGF (e.g., human PDGF-CC), human amphiregulin, and human TGF-α.

[0110] EGF EGF is a potent mitogen for a variety of ectodermal and mesodermal cell cultures and has profound effects on the differentiation of certain cells in vivo and in vitro as well as on the differentiation of some fibroblasts in cell culture. The EGF precursor exists as a membrane-bound molecule that is proteolytically cleaved to yield a 53 amino acid peptide hormone that stimulates cells.

[0111] The present inventors have found that EGF is not essential for the culture medium of the present invention to grow breast organoid.Therefore, in some embodiments, the culture medium of the present invention does not contain EGF.However, the present inventors have found that the addition of EGF may be beneficial for the growth of breast organoid.Therefore, in some embodiments, the culture medium of the present invention further comprises EGF.

[0112] In an embodiment in which the culture medium of the present invention contains EGF, EGF is preferably added to the basal culture medium at a concentration of 1 to 500 ng / ml, or at least 1 ng / ml, but not exceeding 500 ng / ml. Preferred concentrations are at least 1 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 25 ng / ml, 30 ng / ml, 40 ng / ml, 45 ng / ml, or 50 ng / ml, but not exceeding 500 ng / ml, 450 ng / ml, 400 ng / ml, 350 ng / ml, 300 ng / ml, 250 ng / ml, 200 ng / ml, 150 ng / ml, or 100 ng / ml. More preferred concentrations are at least 1 ng / ml, but not exceeding 100 ng / ml. The same concentrations can be used for different mitogenic growth factors, e.g., PDGF or FGF. When multiple FGFs are used, for example FGF7 and FGF10, the concentration of FGFs is as defined above and refers to the total concentration of FGFs used.

[0113] FGFR2b Ligand FGF7 and FGF10 are members of the fibroblast growth factor (FGF) protein family. FGF family members have broad mitogenic and cell survival activities and are involved in a variety of biological processes, including embryonic development, cell growth, morphogenesis, tissue repair, tumor growth, and invasion. FGFs stimulate cells by interacting with cell surface tyrosine kinase receptors (FGFRs). Four closely related receptors (FGFR1-FGFR4) have been identified. The FGFR1-FGFR3 genes have been shown to encode multiple isoforms, and these isoforms may be important in determining ligand specificity. Most FGFs bind to multiple receptors (Ornitz J Biol Chem. 1998 Feb 27;273 (9):5349-57). However, FGF10 and FGF7 are unique among FGFs because they interact only with a specific isoform of FGFR2, called FGFR2b, which is expressed only in epithelial cells (Igarashi, J Biol Chem. 1998 273(21): 13230-5).

[0114] In a preferred embodiment, the culture medium of the present invention comprises an FGFR2b ligand (e.g., FGF1, FGF3, FGF7, FGF10, or FGF22). In some embodiments, the FGFR2b ligand is a high affinity FGFR2b ligand. In some embodiments, the FGFR2b ligand binds to FGFR2b but does not bind to any other FGFR isoforms. In some embodiments, the FGFR2b ligand is a member of the FGF7 subfamily (e.g., FGF7, FGF10, or FGF22). In a preferred embodiment, the FGFR2b ligand is FGF10 and / or FGF7. In some embodiments, multiple FGFR2b ligands are not used. In other embodiments, more than one FGFR2b ligand is used, for example, two, three, or more FGFR2b ligands. In some embodiments, the culture medium comprises FGF7 and FGF10. In some embodiments, the FGFR2b ligand is a biologically active fragment or variant of a natural FGFR2b ligand, for example, a biologically active fragment or variant of FGF7 and / or FGF10.In some embodiments, the FGFR2b ligand in the culture medium of the present invention is a biologically active synthetic ligand. "Biologically active" as used in this context means that an FGFR2b ligand, e.g., a fragment or variant of a native FGFR2b ligand or a synthetic FGFR2b ligand, is capable of binding to FGFR2b and inducing similar downstream signaling compared to FGF7 and / or FGF10, e.g., inducing tyrosine phosphorylation of FRS2α (FGF receptor substrate 2α) or FRS2β (FGF receptor substrate 2β). Tyrosine phosphorylation of FRS2α and / or FRS2β is the earliest cytosolic event in the signaling pathway activated by FGF7 and / or FGF10. In some embodiments, the FGFR2b ligand is substituted with a compound that activates the FGFR2 or FGFR4 pathway (an "FGF pathway activator").

[0115] In some embodiments, the one or more FGFR2b ligands used in the culture medium of the present invention are selected from the following: human FGF7, human FGF10, human FGF22, human FGF1, or human FGF22.

[0116] In further embodiments, a combination of mitogenic growth factors, such as EGF and FGF7, or EGF and FGF10, is added to the basic culture medium. In further preferred embodiments, a combination of mitogenic growth factors, such as (i) FGF7 and FGF10, (ii) EGF, FGF7, and FGF10, or (iii) PDGF, FGF7, and FGF10, or (iv) EGF, PDGF, amphiregulin, FGF7, and FGF10, is added to the culture medium.

[0117] In some embodiments, TGF-α or amphiregulin is added to the basal culture medium. These mitogenic growth factors may replace EGF. In some embodiments, hepatocyte growth factor (HGF) is added to the culture medium.

[0118] During the cultivation of stem cells, preferably, a combination of receptor tyrosine kinase ligands (such as EGF, FGF10 and FGF7) is added to the culture medium when necessary, for example, every day or every other day. They can be added alone or in combination. They are preferably added every other day.

[0119] ROCK inhibitors Any of the described culture media can contain a ROCK inhibitor, for example, Y-27632 (10 μM; Sigma), especially during the first few days of culture before performing cell sorting experiments. Because ROCK inhibitors are known to prevent anoikis (a type of programmed cell death induced by anchorage-dependent cells detached from the surrounding extracellular matrix). Therefore, any of the culture media defined herein can further contain a ROCK inhibitor during the first few days. In some embodiments, the culture medium of the present invention further contains a ROCK inhibitor, for example, Y-27632, during the first few days of culture before performing cell sorting experiments.

[0120] A further embodiment of the culture medium of the present invention comprises a Rock (Rho-kinase) inhibitor. The addition of a Rock inhibitor has been found to prevent anoikis, especially when culturing single stem cells. The Rock inhibitor is preferably selected from R-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632, Sigma-Aldrich), 5-(1,4-diazepan-1-ylsulfonyl)isoquinoline (Fasudil or HA1077, Cayman Chemical), and (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride (H-1152, Tocris Bioscience). The Rho-kinase inhibitor, for example, Y-27632, is preferably added to the culture medium every other day for the first 7 days of culturing the stem cells. The Rock inhibitor is preferably included in the medium for the first few days, for example, the first 1, 2, 3, 4, 5, 6, or 7 days of culture after seeding or splitting one cell. Any suitable Rock inhibitor concentration can be used, for example, 1-200uM, 1-100uM, 5-50uM, or about 10uM. The preferred Y27632 concentration is 10uM. Thus, in some embodiments, the present invention provides a method for culturing stem cells and / or a method for obtaining organoids, in which the Rock inhibitor is added to the culture medium for the first 1, 2, 3, 4, 5, 6, or 7 days, optionally every other day. In some embodiments, no Rock inhibitors are added to the culture medium after the first 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.

[0121] Addition of Rock inhibitor is particularly important when single stem cell is cultured (as mentioned above), i.e., when the starting material of organoid is single stem cell.Therefore, in some embodiments, the present invention provides the method for obtaining organoid, comprising the step of culturing stem cell, optionally single stem cell, and Rock inhibitor is added to culture medium for the first 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days, optionally every other day, and optionally Rock inhibitor is not added to culture medium after the first 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days.

[0122] Rock inhibitor is less important and sometimes not necessary when culturing multiple cells, for example, when the starting material of organoid is tissue fragment.Therefore, in some embodiments, the present invention provides the method for obtaining organoid, comprising the step of culturing stem cells, optionally tissue fragment, and Rock inhibitor is not added to culture medium at all, or is not added to culture medium after the first 2, 3, 4, 5, 6, 7, 8, 9 or 10 days.

[0123] When cells are split into multiple cultures, Rock inhibitor can be added to culture medium in the same way.This means that after splitting, especially when splitting involves taking single stem cells from first culture and putting them into second culture, Rock inhibitor is added for the first 1, 2, 3, 4, 5, 6, or 7 days, optionally every other day.If splitting involves taking multiple stem cells from first culture and putting them into second culture, Rock inhibitor is less important and sometimes not necessary to add.Therefore, in some embodiments, when the method for obtaining organoid or the method for culturing stem cells involves splitting, optionally when single cells are involved in splitting, Rock inhibitor is added to new culture medium for the first 1, 2, 3, 4, 5, 6, or 7 days, optionally every other day after splitting. In some embodiments, when the method for obtaining organoids or the method for culturing stem cells involves splitting, optionally when multiple cells are involved in splitting, they are not added to the culture medium at all, or are not added to the culture medium after the first 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.

[0124] Notch agonists In yet a further embodiment, the culture medium of the present invention further comprises a Notch agonist. Notch signaling has been shown to play an important role in cell fate determination and cell survival and proliferation. Notch receptor proteins can interact with a number of surface-bound or secreted ligands, including, but not limited to, Delta1, Jagged1 and 2, and Delta-like1, Delta-like3, Delta-like4. Upon ligand binding, the Notch receptor is activated by successive cleavage events involving members of the ADAM protease family, as well as intramembrane cleavage regulated by gamma-secretase presenilin. The result is the translocation of the Notch intracellular domain to the nucleus, where it activates the transcription of downstream genes. The preferred Notch agonist is selected from Jagged1 and Delta1, or active fragments or derivatives thereof. The most preferred Notch agonist is a Notch receptor having the sequence The DSL peptide has the structure TIFF0007602446000003.tif4128 (Dontu et al., 2004. Breast Cancer Res 6. R605-R615). The DSL peptide is preferably used at a concentration of 10 μM to 100 nM, or at least 10 μM, but not more than 100 nM. Addition of Notch agonist increases the culture efficiency by 2-3 times, especially when Notch agonist is added in the first week of culture. The Notch agonist is preferably added to the culture medium every other day for the first 7 days of culturing the stem cells. Thus, in some embodiments, the present invention provides a method for culturing stem cells and / or a method for obtaining organoids, in which Notch agonist is added to the culture medium every other day for the first 1, 2, 3, 4, 5, 6, or 7 days, optionally. In some embodiments, a Notch agonist is not added to the culture medium after the first 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.

[0125] A Notch agonist is defined as a molecule that stimulates Notch activity in a cell 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 at least 100%, compared to the level of Notch activity in the absence of said molecule. As known to those skilled in the art, Notch activity can be confirmed by measuring Notch transcription activity, for example, by using a 4xwtCBF1-luciferase reporter construct as described in (Hsieh et al, 1996 Mol Cell. Biol. 16, 952-959).

[0126] Further ingredients The culture medium optionally contains nicotinamide, and is 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 culture medium further contains one or more components selected from the group consisting of nicotinamide, B27, N2, and N-acetylcysteine. For example, in some embodiments, the culture medium further contains B27, N-acetylcysteine, and nicotinamide.

[0127] B27 (Invitrogen), N-acetylcysteine ​​(Sigma) and N2 (Invitrogen), and nicotinamide (Sigma) are believed to regulate cell proliferation and aid in DNA stability. In the context of the present invention, nicotinamide is also referred to herein as "Nic."

[0128] In some embodiments, nicotinamide is present at 7-15 mM, for example, about 10 mM.

[0129] In some embodiments, the B27 supplement is "B27 Supplement minus Vitamin A" (available from Invitrogen, Carlsbad, CA; www.invitrogen.com; currently catalog number 12587010; and PAA Laboratories GmbH, Pasching, Austria; www.paa.com; catalog number F01-002; Brewer et al., J Neurosci Res., 35(5):567-76, 1993) and may be used to formulate culture media that includes biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinyl acetate, sodium selenite, triiodothyronine (T3), DL-alpha tocopherol (vitamin E), albumin, insulin, and transferrin.

[0130] B27 supplement, supplied by PAA Laboratories GmbH, occurs as a 50x liquid concentrate containing, among other components, biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinol, retinyl acetate, sodium selenite, triiodothyronine (T3), DL-α tocopherol (vitamin E), albumin, insulin, and transferrin. Of these components, at least linoleic acid, retinol, retinyl acetate, and triiodothyronine (T3) are nuclear hormone receptor agonists. B27 supplement may be added to the culture medium as a concentrate or may be diluted before being added to the culture medium. B27 supplement may be used at 1x final concentration or other final concentrations. The use of B27 supplement is a convenient way to incorporate biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinol, retinyl acetate, sodium selenite, triiodothyronine (T3), DL-α tocopherol (vitamin E), albumin, insulin, and transferrin into the culture medium of the present invention.Instead of using B27 supplement, it is also envisioned that some or all of these components can be added separately to the culture medium.Therefore, the culture medium can include some or all of these components.

[0131] In some embodiments, retinoic acid is absent from the B27 supplement used in the culture medium and / or is absent from the culture medium.

[0132] "N2 supplement" is available from Invitrogen, Carlsbad, CA; www.invitrogen.com; catalog number 17502-048; and PAA Laboratories GmbH, Pasching, Austria; www.paa.com; catalog number F005-004; Bottenstein & Sato, PNAS, 76(1):514-517, 1979. N2 supplement provided by PAA Laboratories GmbH comes as a 100x liquid 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 the culture medium as a concentrate or may be diluted before being added to the culture medium. N2 supplement may be used at 1x final concentration or other final concentrations. The use of N2 supplement is a convenient way to incorporate transferrin, insulin, progesterone, putrescine and sodium selenite into the culture medium of the present invention.Of course, it is also envisioned that instead of using N2 supplement, some or all of these components can be added separately to the culture medium.Therefore, the culture medium can include some or all of these components.

[0133] In some embodiments in which the medium contains B27, the medium also does not contain N2. Thus, when B27 is present, embodiments of the invention can be adapted to exclude N2, if desired.

[0134] In some embodiments, N2 is absent from the culture medium.

[0135] In some embodiments where the medium contains N2, the medium also does not contain B27. Thus, when N2 is present, embodiments of the invention can be adapted to exclude B27, if desired.

[0136] In some embodiments, B27 is absent from the culture medium.

[0137] In some embodiments, the culture medium is supplemented with B27 and / or N2.

[0138] In some embodiments, the basal medium is supplemented with 150 ng / ml to 250 ng / ml of N-acetylcysteine.Preferably, the basal medium is supplemented with about 200 ng / ml of N-acetylcysteine.

[0139] In some embodiments, the culture medium contains a progestin. In other embodiments, the culture medium does not contain a progestin.

[0140] In some embodiments, the culture medium comprises estradiol. In other embodiments, the culture medium does not comprise estradiol.

[0141] In some embodiments, the culture medium comprises hepatocyte growth factor. In other embodiments, the culture medium does not comprise hepatocyte growth factor.

[0142] In some embodiments, the culture medium contains receptor activator of nuclear factor kappa-B ligand (RANKL). In other embodiments, the culture medium does not contain RANKL. In some embodiments, nicotinamide is added to the culture medium of the present invention. The addition of nicotinamide has been found to improve the culture efficiency and lifespan of human organoids. Nicotinamide may be added to the culture medium to a final concentration of 1 to 100 mM, 5 to 50 mM, or preferably 5 to 20 mM. For example, nicotinamide may be added to the culture medium to a final concentration of about 10 mM.

[0143] In some embodiments, the culture medium of the present invention comprises a p53 stabilizer. To ensure that the cell population is mainly tumor cells, a p53 stabilizer may be added to the culture medium. Without intending to be bound by any theory, it is believed that the p53 stabilizer increases the cellular concentration of p53 (e.g., by blocking the interaction between p53 and Mdm2), stimulates p53-dependent expression, and induces cellular senescence. p53 mutations usually occur in tumor cells, which may result in mutant p53 proteins that cannot be stabilized by p53 stabilizers. Thus, it is believed that a subset of tumor cells with mutant p53 proteins escapes from p53 stabilizer-induced senescence and grows faster than normal cells in a mixed population. A preferred p53 stabilizer is a member of the Nutlin family, such as Nutlin-1, Nutlin-2, or Nutlin-3. For example, in some embodiments, the p53 stabilizer is Nutlin-3. Any suitable concentration of p53 stabilizer can be used, for example, 1 nM to 10 mM, 10 nM to 1 mM, 100 nM to 100 μM, 1 μM to 10 μM. For example, Nutlin-3 may be added to the culture medium of the invention to a final concentration of about 5 μM. Thus, in some embodiments of the methods of the invention where mammary epithelial stem cells are cancer cells, the culture medium used in the method advantageously comprises a p53 stabilizer. However, other embodiments of such methods for culturing mammary epithelial cancer stem cells do not use a culture medium comprising a p53 stabilizer. It is also envisioned that when the method is used to culture normal mammary epithelial stem cells, but a p53 stabilizer is not required, the p53 stabilizer may not be present in the culture medium.

[0144] In some embodiments, the culture medium of the present invention comprises a p38 MAP kinase inhibitor. The p38 MAP kinase inhibitor is an inhibitor that directly or indirectly negatively regulates p38 signal transduction. In some embodiments, the p38 MAP kinase inhibitor is SB202190. However, other suitable p38 MAP kinase inhibitors may be used instead, and these are easily available to those skilled in the art.

[0145] Any suitable pH can be used. For example, the pH of the medium may be within the range of about 7.0 to 7.8, within the range of about 7.2 to 7.6, or about 7.4. The pH may be maintained using a buffer. Suitable buffers can be readily selected by one of skill in the art. Buffers that may be used include carbonate buffers (e.g., NaHCO 3 ) and phosphate buffer (e.g., NaH 2 PO 4 ) are included. These buffers are typically 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) can also be used, typically at about 1000 to about 10,000 mg / L. The culture medium may contain a pH indicator such as phenol red (e.g., about 5 to about 50 mg / L) to facilitate monitoring of the pH status of the medium.

[0146] The culture medium for use 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 stem cell culture medium.The amino acids that can 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 culture medium contains all of these amino acids. Generally, with the exception of L-glutamine, which is present at about 0.05 to about 1 g / L (usually about 0.1 to about 0.75 g / L), each amino acid, when present, is present at about 0.001 to about 1 g / L medium (usually about 0.01 to about 0.15 g / L). The amino acids may be of synthetic origin.

[0147] The culture medium for use 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 stem cell culture medium.Vitamins that may be present include thiamine (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), D-calcium pantothenate (vitamin B5), pyridoxal / pyridoxamine / pyridoxine (vitamin B6), folic acid (vitamin B9), cyanocobalamin (vitamin B12), ascorbic acid (vitamin C), calciferol (vitamin D2), DL-α tocopherol (vitamin E), biotin (vitamin H), and menadione (vitamin K).

[0148] The culture medium for use in the present invention may contain one or more inorganic salts. Those skilled in the art will understand the appropriate type and amount of inorganic salts to use in stem cell culture medium. Inorganic salts are typically included in culture medium to help maintain the osmotic balance of the cells and to help regulate the membrane potential. Inorganic salts that may be present include calcium, copper, iron, magnesium, potassium, sodium, and zinc salts. Salts are usually used in the form of chlorides, phosphates, sulfates, nitrates, and bicarbonates. Specific salts that may be used include CaCl 2 , CuSO 4 -5H 2 O, Fe(NO 3 )-9H 2 O, FeSO 4 -7H 2 O, MgCl, MgSO 4 , KCl, NaHCO 3 , NaCl, Na 2 HPO 4 , Na 2 HPO 4 -H 2 O, and ZnSO 4 -7H 2 Contains O.

[0149] The osmolarity of the medium may 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 may be less than about 300 mOsm / kg (e.g., about 280 mOsm / kg).

[0150] The culture medium for use 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 type and amount of sugar to use in stem cell culture 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 is typically present at about 1 to about 10 g / L.

[0151] The culture 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% by volume of medium according to conventional techniques.

[0152] In another embodiment, the culture medium of the present invention may contain serum substitute. A variety of different serum substitute formulations are commercially available and known to those skilled in the art. When serum substitute is used, it may be used at about 1% to about 30% by volume of medium according to conventional techniques.

[0153] In other embodiments, the culture medium of the present invention may be serum-free and / or serum substitute-free. Serum-free medium is a medium that does not contain any type of animal serum. Serum-free medium may be preferred to avoid possible xeno-contamination of stem cells. Serum-substitute-free medium is a medium that is not supplemented with any commercially available serum substitute preparation.

[0154] In preferred embodiments, cell culture medium is supplemented with purified growth factors, natural growth factors, semi-synthetic growth factors, and / or synthetic growth factors, and does not contain unknown 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, cell culture medium is supplemented with one or more of these supplements, for example, one, any two, or all three of these supplements.

[0155] Culture media for use in the present invention may contain one or more trace elements, such as ions of barium, bromium, cobalt, iodine, manganese, chromium, copper, nickel, selenium, vanadium, titanium, germanium, molybdenum, silicon, iron, fluorine, silver, rubidium, tin, zirconium, cadmium, zinc, and / or aluminum.

[0156] The medium may also contain a reducing agent, for example, β-mercaptoethanol at a concentration of about 0.1 mM.

[0157] The culture medium of the present invention may contain one or more additional agents, such as nutrients or growth factors that have been reported to improve stem cell culture, such as cholesterol / transferrin / albumin / insulin / progesterone, putrescine, selenite / other factors.

[0158] In some embodiments, the culture medium of the present invention comprises recombinant human neuregulin beta-1, Rspondin (e.g., Rspondin1), Noggin, B27, N-acetylcysteine, nicotinamide, ROCK1,2 inhibitors (e.g., Y-27632), ALK4,5,7 inhibitors (e.g., A83-01), p38 MAP kinase inhibitors (e.g., SB202190), FGF7, and FGF10.

[0159] In some embodiments, the culture medium of the present invention comprises recombinant human neuregulin beta-1, Rspondin (e.g., Rspondin1), Noggin, B27, N-acetylcysteine, nicotinamide, ROCK1,2 inhibitors (e.g., Y-27632), ALK4,5,7 inhibitors (e.g., A83-01), p38 MAP kinase inhibitors (e.g., SB202190), FGF7, FGF10, and PDGF-CC.

[0160] In some embodiments, the culture medium of the present invention comprises recombinant human neuregulin beta-1, Rspondin (e.g., Rspondin1), Noggin, B27, N-acetylcysteine, nicotinamide, ROCK1,2 inhibitor (e.g., Y-27632), ALK4,5,7 inhibitor (e.g., A83-01), p38 MAP kinase inhibitor (e.g., SB202190), FGF7, FGF10, and EGF.

[0161] In some embodiments, the culture medium of the present invention comprises recombinant human neuregulin beta-1, Rspondin (e.g., Rspondin1), Noggin, B27, N-acetylcysteine, nicotinamide, ROCK1,2 inhibitors (e.g., Y-27632), ALK4,5,7 inhibitors (e.g., A83-01), p38 MAP kinase inhibitors (e.g., SB202190), FGF7, FGF10, amphiregulin, TGF-α, and EGF.

[0162] Extracellular matrix As described above, the method for culturing epithelial stem cells comprises culturing one or more epithelial stem cells in contact with extracellular matrix.Any suitable extracellular matrix can be used.The isolated epithelial stem cells are preferably cultured in a microenvironment that at least partially mimics the cell niche in which the stem cells naturally exist.This cell niche can be mimicked by culturing the stem cells in the presence of a biomaterial, for example, an extracellular matrix that provides important regulatory signals that control stem cell fate.

[0163] Cell niche is determined in part by stem cells and surrounding cells, and the extracellular matrix (ECM) produced by cells in said niche.In the preferred method of the present invention, epithelial stem cells are cultured in contact with ECM.By "in contact" is meant physical or mechanical or chemical contact.By physical or mechanical or chemical contact, it is meant that force must be used to separate the resulting organoid or epithelial stem cell population from the extracellular matrix.Preferably, epithelial stem cells are embedded in ECM.

[0164] The culture medium of the present invention may be dispersed in an extracellular matrix (ECM). In a preferred method of the present invention, the isolated tissue fragment or isolated epithelial stem cell is attached to the ECM. The ECM is composed of various polysaccharides, water, elastin, and glycoproteins, including collagen, entactin (nidogen), fibronectin, and laminin. The ECM is secreted by epithelial cells, endothelial cells, parietal endoderm-like cells (e.g., Englebreth-Holm-Swarm parietal endoderm-like cells as described in Hayashi et al. (2004) Matrix Biology 23:47-62), and connective tissue cells. Various types of ECM are known, including various compositions including various types of glycoproteins and / or various combinations of glycoproteins. The ECM can be provided by culturing ECM-producing cells, such as epithelial cells, endothelial cells, parietal endoderm-like cells, or fibroblast cells in a container, removing these cells, and adding the isolated tissue fragment or isolated epithelial stem cell. Examples of extracellular matrix producing cells are 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. Alternatively, the ECM is commercially sourced. Examples of commercially available extracellular matrices are extracellular matrix proteins (Invitrogen), basement membrane preparations from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells (e.g., Cultrex® Basement Membrane Extract (Trevigen, Inc.), or Matrigel™ (BD Biosciences)). Synthetic extracellular matrix materials, such as ProNectin (Sigma Z378666), may also be used. If desired, mixtures of extracellular matrix materials may also be used. The use of ECM to culture stem cells extended the long-term survival of stem cells and enhanced the continued presence of undifferentiated stem cells.In the absence of ECM, stem cell cultures could not be cultured for long periods of time, and no continued presence of undifferentiated stem cells was observed. Furthermore, in the presence of ECM, it was possible to culture three-dimensional tissue organoids, which cannot be cultured in the absence of ECM. The extracellular matrix material is usually sunk to the bottom of the dish in which the cells are suspended. Typically, when the matrix solidifies at 37°C, medium is added and diffuses into the ECM. The cells in the medium anchor to the ECM by interacting with the surface structure of the ECM, for example, by interacting with integrins.

[0165] One example of an ECM for use in the methods of the present invention includes at least one glycoprotein, such as laminin.

[0166] A preferred ECM for use in the method of the present invention comprises at least two different glycoproteins, such as two different types of collagen, or collagen and laminin. The ECM may be a synthetic hydrogel extracellular matrix or a natural ECM. A more preferred ECM comprises laminin, entactin, and collagen IV. A more preferred ECM is provided by Matrigel™ (BD Biosciences), which comprises laminin, entactin, and collagen IV. In some embodiments, the extracellular matrix is ​​a laminin-containing extracellular matrix, such as Matrigel™ (BD Biosciences). In some embodiments, the ECM comprises laminin, entactin, collagen IV, and heparin sulfate proteoglycan (e.g., Cultrex® Basement Membrane Extract Type 2 (Trevigen, Inc.)).

[0167] In some embodiments, single stem cells, cell populations, or tissue fragments are embedded in Matrigel, which is optionally low in growth factors and / or phenol red free.

[0168] In some embodiments, culture medium is placed on top of ECM. Culture medium can then be removed and replenished as and when necessary. In some embodiments, culture medium is replenished every 1 day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, or every 7 days. If a component is "added" or "removed" from medium, this means that in some embodiments, the medium itself is removed from ECM, and then new medium containing the "added" component or new medium excluding the "removed" component is placed on ECM.

[0169] In some embodiments, the culture medium of the present invention is in contact with an extracellular matrix or a 3D matrix that mimics the extracellular matrix by interacting with cell membrane proteins, such as integrins.

[0170] Additionally, there is provided a culture medium and extracellular matrix of the invention, eg, supplied as a kit.

[0171] In some embodiments, the culture medium of the present invention is suitable for culturing adult breast stem cell.In some embodiments, the culture medium of the present invention is suitable for obtaining breast organoid.In some embodiments, the culture medium of the present invention is suitable for expanding breast stem cell population for at least 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th or more than 14th passages.

[0172] Thus, the present invention provides culture media as described herein comprising one or more ErbB3 / 4 ligands suitable for expanding a mammary stem cell population for at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more passages.

[0173] In some embodiments, the culture medium of the present invention is suitable for establishing the successful population of breast organoids in at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of inoculations.Successful breast organoids are defined herein as the organoids that can be passaged at least 4 times.

[0174] Thus, the present invention provides culture media comprising one or more ErbB3 / 4 ligands suitable for establishing successful populations of organoids with an efficiency of at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0175] Uses of the culture medium of the present invention The invention provides the use of a culture medium of the invention for expanding epithelial stem cells, epithelial stem cell populations, tissue fragments, or organoids.

[0176] The present invention also provides a use of the culture medium of the present invention for expanding mammary epithelial stem cells, mammary epithelial stem cell populations, mammary tissue fragments, or mammary organoids.

[0177] In some embodiments, stem cells, stem cell populations, tissue fragments or organoids can be obtained from normal tissue.For example, in some embodiments, stem cells, stem cell populations, tissue fragments or organoids can be obtained from non-cancerous tissue.

[0178] In some embodiments, stem cell, stem cell population, tissue fragment, or organoid can be obtained from disease tissue, for example, cancer tissue, for example, adenoma, fibroadenoma, adenocarcinoma, carcinoma, sarcoma, circulating tumor cell, or metastasized cancer.Thus, in some embodiments, stem cell, stem cell population, tissue fragment, or organoid can be obtained from benign breast tumor or malignant breast tumor.In some embodiments, stem cell, stem cell population, tissue fragment, or organoid is or comprises breast cancer cell.In some embodiments, stem cell, stem cell population, or tissue fragment is a biopsy material from breast tumor.Said tumor is preferably the original breast tumor, not the metastatic tumor that arises from different tissue.

[0179] Isolation of Mammary Epithelial Stem Cells for Culture In a preferred embodiment, the epithelial stem cell that is cultured in the method of the present invention and / or that obtains organoid is obtained from adult tissue.That is, the epithelial stem cell is adult epithelial stem cell.In this context, "adult" refers to mature tissue, that is, includes newborn or child, but excludes embryo or fetus.In a preferred embodiment, the epithelial stem cell is not derived from embryonic stem cell or embryonic stem cell line, for example, not derived from embryonic stem cell or embryonic stem cell line that is differentiated in vitro, for example, not derived from human embryonic stem cell or human embryonic stem cell line.

[0180] The cells and tissues are preferably mammalian cells and tissues.More preferably, the cells and tissues are human cells and tissues.In some embodiments, the cells and tissues are derived from other mammals, such as laboratory animals (e.g., mice, rabbits, rats, guinea pigs), companion animals (e.g., dogs, cats, horses), or livestock (e.g., cows, pigs, sheep, goats).

[0181] Cells taken directly from living tissue, i.e., freshly isolated cells, are also called primary cells. In some embodiments, the epithelial stem cells are primary epithelial stem cells. Primary cells are the best experimental model of the in vivo situation. In a preferred embodiment of the present invention, the epithelial stem cells are primary epithelial stem cells (or are derived from primary epithelial stem cells). Primary cell cultures can be passaged to form secondary cell cultures. Except for cancer cells, traditional secondary cell cultures have a limited life span. After a certain number of population doublings (e.g., 50-100 generations), the cells undergo a senescence process and stop dividing. Cells derived from the secondary cultures can be immortalized to become continuous cell lines. Immortalization can occur naturally or can be induced virally or chemically. Immortalized cell lines are also known as transformed cells. In contrast, the methods of the present invention allow for continuous passage of epithelial stem cells without immortalization or transformation. Thus, in some embodiments, the epithelial stem cells are not immortalized or transformed cells, and are not derived from immortalized or transformed cell lines. An advantage of the present invention is that epithelial stem cells that have undergone multiple rounds of expansion and passage retain the characteristics of primary cells and have minimal or no genotypic or phenotypic changes.

[0182] Epithelial stem cells can be obtained by any suitable method. In some embodiments, cells are isolated by collagenase digestion, for example, as described in the Examples and Dorell et al., 2008 (Hepatology. 2008 Oct;48(4): 1282-91. Surface markers for the murine oval cell response. Dorrell C, Erker L, Lanxon-Cookson KM, Abraham SL, Victoroff T, Ro S, Canaday PS, Streeter PR, Grompe M). In some embodiments, collagenase digestion is performed on tissue biopsy. In some embodiments, collagenase digestion and accutase digestion are used to obtain epithelial stem cells for use in the present invention.

[0183] In some embodiments, breast tissue may be minced, washed (e.g., with basal culture medium), and incubated in a culture medium of the invention supplemented with a protease (e.g., collagenase, dispase, or trypsin) (see, e.g., Example 1). The protease may be at any suitable concentration, e.g., 0.1-10 mg / ml, 0.1-1 mg / ml, or 1-10 mg / ml. For example, the protease may be present at 1-2 mg / ml. Incubation in culture medium supplemented with the protease may be for any suitable length of time. For example, the tissue may be incubated in the medium for 10 minutes to 10 hours, 20 minutes to 5 hours, 30 minutes to 5 hours, 30 minutes to 3 hours, 30 minutes to 2 hours, 1 hour to 5 hours, 1 hour to 3 hours, or 1 hour to 2 hours. In some embodiments, the tissue is incubated in the medium for at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, or at least 5 hours.

[0184] The tissue fragments contained in the resulting digested tissue suspension may then be further reduced in size. For example, the suspension may be sheared. Filtration and / or centrifugation steps may be used to isolate the tissue fragments or isolated cells. The isolated tissue fragments or isolated cells may then be cultured in the culture medium of the present invention.

[0185] In some embodiments, the method comprises culturing a fragment of tissue comprising the epithelium, hi some embodiments, epithelial stem cells are isolated from the tissue fragment.

[0186] In some embodiments, epithelial stem cells are obtained for culture based on the expression of epithelial cell surface markers, such as EPCAM, MUC-1, KRT-5, KRT8, and KRT18. Thus, the method of the present invention includes the steps of preparing a cell suspension from the epithelial tissue as described above, contacting the cell suspension with an epithelial cell surface marker binding compound (e.g., EPCAM, MUC-1, KRT-5, KRT8, and / or KRT18 binding compound), isolating the epithelial marker binding compound, and isolating the cells from the binding compound.

[0187] In some embodiments, epithelial stem cells are obtained for culturing based on the expression of Lgr5 and / or Lgr6 on the surface of epithelial stem cells. These proteins belong to the large G protein-coupled receptor (GPCR) superfamily (see, for example, WO2009 / 022907, the contents of which are incorporated herein in their entirety). The Lgr subfamily is unique in that it carries a large leucine-rich ectodomain that is important for ligand binding. Thus, in some embodiments, the method of the present invention includes the steps of preparing a cell suspension from the epithelial tissue as described above, contacting the cell suspension with a compound that binds to Lgr5 and / or Lgr6 (e.g., an antibody, e.g., an anti-Lgr5 monoclonal antibody, e.g., an anti-Lgr5 monoclonal antibody described in WO2009 / 022907), isolating the compound that binds to Lgr5 and / or Lgr6, and isolating stem cells from the binding compound.

[0188] The organoids are preferably obtained using cells derived from adult tissue, preferably epithelial stem cells derived from adult tissue.

[0189] In some embodiments, epithelial stem cell is normal cell.In alternative embodiments, epithelial stem cell is cancer stem cell.Therefore, it is envisaged that stem cell can be Lgr5 positive cancer stem cell, for example.Therefore, said cell can be obtained from tumor, if necessary.In alternative embodiments, epithelial stem cell is diseased stem cell, for example, stem cell infected with intracellular pathogen (for example, bacteria, virus or parasite).

[0190] A binding compound that binds to breast cancer cell markers may be used to isolate breast cancer stem cells for culturing in the method of the present invention. Examples of breast cancer cell markers include epithelial membrane antigen, cancer antigen 15-3 (CA15-3), cancer antigen 27.29 (CA27.29), carcinoembryonic antigen (CEA), urokinase plasminogen activator (uPA), plasminogen activator inhibitor (PAI-1), podocalyxin, EZH2, and kallikrein-10. For example, an epithelial membrane antigen (EMA) binding compound (e.g., anti-EMA antibody) may be used to isolate breast cancer cells. Thus, in some embodiments, the method of the present invention includes the steps of preparing a cell suspension from primary or secondary cancer tissue, contacting the cell suspension with a breast cancer cell marker binding compound (e.g., EMA binding compound), isolating the breast cancer cell marker binding compound, and isolating cells from the binding compound. In another embodiment, organoid is generated from single cell, optionally expressing Lgr5 single cell.In some embodiments, single cell comprises the nucleic acid construct that comprises the nucleic acid molecule of interest.

[0191] Preferred Lgr5 and / or Lgr6 binding compounds include antibodies that specifically recognize and bind to the extracellular domain of either Lgr5 or Lgr6, such as monoclonal antibodies, such as monoclonal antibodies including mouse and rat monoclonal antibodies (see, for example, WO2010 / 016766, the contents of which are incorporated herein in their entirety). Using such antibodies, stem cells expressing Lgr5 and / or Lgr6 can be isolated, for example, with the aid of magnetic beads or by fluorescence-activated cell sorting, as will be apparent to those skilled in the art. Using the method of the present invention, it is possible to isolate only one cell expressing Lgr5 and / or Lgr6 and apply the method of the present invention thereto. Thus, organoid or breast epithelial stem cell population can be derived from only one cell. Thus, in some embodiments, the starting cell to be cultured is one cell.

[0192] Alternatively, a cell population may be used as a starting point, for example a cell population contained in a breast tissue fragment as described above. Thus, the methods of the invention are not limited to using a single cell as a starting point.

[0193] In a further aspect, the method for obtaining organoid is provided, comprising culturing mammary epithelial stem cell in the culture medium of the present invention.Preferably, said method comprises culturing mammary epithelial stem cell in the culture medium of the present invention by using the culture method described herein.

[0194] In some embodiments, said method comprises culturing epithelial stem cells or obtaining the organoid / population of adult epithelial stem cells from one cell.Advantageously, this allows homogeneous cell population to form. In some embodiments, the method includes culturing stem cells in a culture medium of the present invention for a period of time, e.g., 3 days to 10 weeks, 1 to 10 weeks, 1 to 4 weeks, or 10 days to 3 weeks, then passaging the cells (e.g., dissociating the cells to a cell density of 1 and seeding one or more cells at a ratio of 1 cell per container (e.g., per well), expanding the cells using the culture medium of the present invention for a period of time, e.g., 3 days to 10 weeks, 1 to 10 weeks, 1 to 4 weeks, or 10 days to 3 weeks, and repeating the passaging and expanding steps at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, or at least 14 times.

[0195] After culturing, the method may further comprise obtaining and / or isolating one or more epithelial stem cells or organoids. For example, after culturing stem cells, it may be useful to remove one or more stem cells and / or one or more lung organoids cultured in culture medium from the culture medium for use in later applications. To select the cells of the present invention and distinguish these cells from other cell types, any one of a number of physical separation methods known in the art can be used. Such physical methods may involve FACS and various immunoaffinity methods based on the markers that the cells of the present invention specifically express. As described herein, LGR5 is a cell marker that may be expressed in the cells of the present invention. Thus, by way of example only, the cells of the present invention may be isolated by a number of physical separation methods that rely on the presence of this marker. Similarly, any other markers that the cells express can be used.

[0196] In one embodiment, the cells of the present invention may be isolated by FACS using an antibody, for example, an antibody against one of these markers. As will be clear to those skilled in the art, this may be achieved by a fluorescently labeled antibody or a fluorescently labeled secondary antibody with binding specificity to the primary antibody. Examples of suitable fluorescent labels include, but are not limited to, FITC, Alexa Fluor® 488, GFP, CFSE, CFDA-SE, DyLight488, PE, PerCP, PE-Alexa Fluor® 700, PE-Cy5 (TRI-COLOR®), PE-Cy5.5, PI, PE-Alexa Fluor® 750, and PE-Cy7. This list is provided by way of example only and is not intended to be limiting.

[0197] It will be clear to those skilled in the art that FACS analysis using anti-Lgr5 antibody can obtain purified cell population.However, in some embodiments, it may be preferable to further purify cell population by carrying out additional rounds of FACS analysis using other identifiable markers, such as one or more of Sox9, Slug, CD44, and / or ALDH1, although other markers may also be used.

[0198] In another embodiment, the cells of the present invention may be isolated by immunoaffinity purification, a separation method well known in the art. By way of example only, the cells of the present invention may be isolated by immunoaffinity purification against c-kit. As will be apparent to those skilled in the art, this method relies on the immobilization of an antibody on a purification column. The cell sample is then loaded onto the column, whereby the appropriate cells bind to the antibody and thus to the column. After a washing step, the cells are eluted from the column using a competitor that preferentially binds to the immobilized anti-c-kit antibody, allowing the cells to be released from the column.

[0199] It will be clear to those skilled in the art that immunoaffinity purification using immobilized antibodies can provide purified cell populations.However, in some embodiments, it may be preferable to further purify the cell population by performing additional rounds of immunoaffinity purification using one or more of other identifiable markers, such as Sox9, Slug, CD44, and / or ALDH1, and use aliquots of isolated clones to confirm the expression of other relevant intracellular markers.

[0200] It will be clear to those skilled in the art that successive purification steps involving the same physical separation method are not necessarily required. Thus, for example, it will be clear that cells can be purified by a FACS step using anti-Lgr5 antibody followed by an immunoaffinity purification step using SSEA-1 affinity column. In certain embodiments, the cells can be cultured for at least about 15 days, at least about 20 days, at least about 25 days, or at least about 30 days after isolation. In certain aspects, the cells are grown in culture for an extended period of time to improve the homogeneity of the cell phenotype of the cell population.

[0201] Other features of the method are defined in the description section intended for definition. As known in the art, single cell suspensions or small cell clusters (2-50 cells / cluster) are usually seeded rather than large cell clusters. Such cells are seeded on the support at a density that promotes cell proliferation when they divide. Typically, when single cells are isolated, a plating density of at least 1-500 cells / well is used, and the surface of the well is less than 0.32 cm. 2 When the clusters are seeded, the plating density is preferably 250-2500 cells / cm. 2 For replating, in some embodiments, about 2500 cells / cm 2 ~about 5,000 cells / cm 2 During replating, as known in the art, typically single cell suspensions or small cell clusters are seeded rather than large cell clusters.

[0202] In one embodiment, the present invention provides one or more organoids comprising cell group or said stem cell, which is produced or obtained by culturing stem cell or tissue fragment according to the present invention.Claim said stem cell or tissue fragment is cultured for at least 2 months, at least 3 months, preferably at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 9 months or at least 12 months or longer, and stem cell is passaged about every 1-4 weeks.

[0203] A "population" of cells is any number of cells greater than one, but preferably at least 1 x 10 3 cells, at least 1x10 4 cells, at least 1x10 5 cells, at least 1x10 6 cells, at least 1x10 7 cells, at least 1x10 8 cells, or at least 1x10 9 Each cell is a single cell.

[0204] The stem or progenitor cells of the invention cultured according to the invention may be human stem or progenitor cells.The stem cells of the invention cultured according to the invention may be epithelial stem or progenitor cells.

[0205] In some embodiments, the stem cells of the present invention and / or the stem cells cultured according to the present invention are not embryonic stem cells. In some embodiments, the stem cells of the present invention and / or the stem cells cultured according to the present invention are not human embryonic stem cells. Preferably, the stem cells of the present invention are adult stem cells.

[0206] In a preferred embodiment, the stem cells are human mammary epithelial stem cells. Human epithelial stem cells include stem cells derived from human epithelial tissue. Epithelial stem cells can form the distinct cell types that make up the epithelium.

[0207] In a preferred embodiment, the organoids are human breast organoids.

[0208] Methods and culture media for cancer cells and cancer organoids In some embodiments, the epithelial stem cells are cancer cells or non-cancerous tumor cells, e.g., derived from a tumor. In some embodiments, the cancer cells are circulating tumor cells. In some embodiments, the cancer cells are not circulating tumor cells. When the term "cancer" is used herein, it should be understood that this term applies equally to non-cancerous (benign) tumors. Thus, in some embodiments, the cells are derived from malignant tumors or metastases. In some embodiments, the cells are derived from benign tumors. Cancer cells tend to have mutations that constitutively activate or inactivate certain growth pathways. This means that certain factors that are normally required for growth and are in the culture medium are no longer required. For example, some cancers constitutively activate the Wnt pathway. In such cases, the culture medium may not require Lgr5 agonists and / or may not require Wnt agonists, although one or both of these may still be present in the culture medium. Other mutations could allow other factors to be removed from the culture medium described herein. Other epithelial cancers (carcinomas) or non-cancerous tumors (e.g., adenomas) can also grow in the culture medium of the present invention.

[0209] In a preferred embodiment, the cancer organoid obtained from cancer stem cells is grown in the culture medium of the present invention suitable for the growth of the corresponding normal tissue organoid obtained from normal stem cells, and this medium is optionally free of certain factors.For example, the breast cancer organoid obtained by culturing breast cancer stem cells can be grown in the same culture conditions as the normal breast organoid obtained by culturing breast epithelial stem cells, and this medium is optionally free of certain factors.In many situations, it may be preferable (or at least more convenient) to grow cancer organoid in normal tissue medium (without removing any factors).Preferably, using normal tissue medium should allow all genetic backgrounds of cancer to grow without removing specific cancer mutations.Therefore, the method of the present invention for culturing stem cells can be carried out.

[0210] In a further preferred embodiment, cancer organoid is cultured in the culture medium of the present invention, which further comprises p53 stabilizer.To ensure that cell population is mainly tumor cell, p53 stabilizer can be added to the culture medium.Therefore, the method of the present invention for culturing stem cell can be carried out.

[0211] method The present invention provides a method for culturing single mammary epithelial stem cells, populations of mammary epithelial stem cells, or isolated mammary tissue fragments, preferably to generate organoids, comprising the steps of: Providing mammary epithelial stem cells, a population of mammary epithelial stem cells, or an isolated breast tissue fragment; providing a culture medium comprising one or more ErbB3 / 4 ligands, preferably further comprising one or more FGFR2b ligands and / or one or more Wnt agonists; contacting the stem cells, stem cell population, or isolated tissue fragment with a culture medium; Culturing the stem cells, stem cell population, or isolated tissue fragment under appropriate conditions.

[0212] The present invention also provides a method for expanding mammary epithelial stem cells, comprising the steps of: Providing mammary epithelial stem cells, a population of mammary epithelial stem cells, or an isolated breast tissue fragment; Providing a culture medium comprising one or more ErbB3 / 4 ligands and (i) one or more FGFR2b ligands and / or (ii) one or more Wnt agonists (e.g., one or more Lgr5 agonists); contacting the stem cells, stem cell population, or isolated tissue fragment with a culture medium; and Culturing the cells, stem cell population, or isolated tissue fragment under appropriate conditions.

[0213] Preferably, the cells are expanded to generate one or more (e.g., at least 2, 3, 4, 5, 6, 10, 15, 20, or more than 20) organoids.

[0214] In some embodiments, the culture medium used in the methods of the invention is a culture medium according to the invention.

[0215] Methods for "expanding" a cell population or isolated tissue fragment are methods that involve maintaining or increasing the number of stem cells in the initial population to yield an expanded stem cell population that retains the undifferentiated phenotype and self-renewal properties.

[0216] However, this method may also include generating differentiation progeny, which may form tissue-like structures, such as those that contribute to organoid formation.Therefore, the present invention provides a method for obtaining breast organoid, comprising culturing stem cells or tissue fragments comprising said stem cells in the culture medium of the present invention.The present invention provides a method for growing single stem cells or stem cell populations, preferably to produce organoids, comprising culturing single breast stem cells or breast stem cell populations in the culture medium of the present invention.As mentioned above, organoids may be normal organoids, cancer organoids, such as carcinoma organoids or adenocarcinoma organoids, and may be benign tumor organoids, for example.In some embodiments, the method for growing single stem cells or stem cell populations, preferably to produce organoids, comprises growing single stem cells, stem cell populations, or tissue fragments in the culture medium of the present invention.

[0217] Thus, the present invention provides a method for expanding single mammary epithelial stem cells or populations of mammary epithelial stem cells, preferably to generate organoids, comprising the steps of: Providing mammary epithelial stem cells, a population of mammary epithelial stem cells, or an isolated breast tissue fragment; Preparing a culture medium according to the invention; contacting the stem cells with a culture medium; Culturing the cells under appropriate conditions.

[0218] In some embodiments, the method includes contacting the stem cells, stem cell population, or isolated tissue fragments and culture medium with an extracellular matrix, or a 3D matrix as described herein, e.g., a 3D matrix that mimics the extracellular matrix by interacting with cell membrane proteins such as integrins, e.g., a laminin-containing extracellular matrix such as Matrigel™ (BD Biosciences) or Cultrex® Basement Membrane Extract (Trevigen, Inc.). In some embodiments, the culture medium is diffused into the extracellular matrix.

[0219] The present invention provides a method for culturing mammary epithelial stem cells.Furthermore, the present invention provides a method for expanding mammary epithelial stem cells and a method for obtaining mammary organoids.

[0220] The term "expanding stem cells" is synonymous with "obtaining breast organoids."

[0221] Isolation and culture of tissue fragments and stem cells A person skilled in the art would be able to determine suitable culture conditions for the methods of the present invention.

[0222] In some embodiments, the method includes contacting the stem cells, stem cell population, or isolated tissue fragments and culture medium with an extracellular matrix, or a 3D matrix as described herein, e.g., a 3D matrix that mimics the extracellular matrix by interacting with cell membrane proteins such as integrins, e.g., a laminin-containing extracellular matrix such as Cultrex® Basement Membrane Extract (Trevigen, Inc.) or Matrigel™ (BD Biosciences). In some embodiments, the culture medium is diffused into the extracellular matrix.

[0223] In some embodiments, the tissue fragments or isolated cells are cultured in a suspension plate. In some embodiments, the tissue fragments or isolated cells are cultured at 35° C. to 39° C. or 36° C. to 38° C. For example, the tissue fragments or isolated cells may be cultured at about 37° C.

[0224] In some embodiments, the tissue fragments or isolated cells are 2 In some embodiments, the cells are cultured at a concentration of 0. 2 Levels can be, for example, 0.5%-4%, 1%-4%, 1%-3%, or 2%-4%. For example, tissue fragments or isolated cells can be incubated at about 2% O 2 Tissue fragments or isolated cells may be cultured under conditions of 3%-7% CO 2 or 4% to 6% CO 2 For example, tissue fragments or isolated cells may be cultured under conditions of about 5% CO 2 The cells may be cultured under the following conditions:

[0225] The culture medium may be changed at any suitable interval, for example, every 1-7 days, every 2-6 days, or every 3-5 days. For example, the culture medium may be changed every 4 days.

[0226] Organoids may be passaged at any suitable interval, for example, every 3 days to every 10 weeks, every 1 to 10 weeks, every 1 to 4 weeks, or every 10 days to 3 weeks. In some embodiments, organoids are passaged about every 10 days to 3 weeks.

[0227] In some embodiments, the timing to passage the organoids is determined by the organoids reaching a certain size (e.g., an average size of about 100-200 µm) and / or experiencing a decrease in viability as manifested by loss of epithelial integrity, the organoids having a dense appearance, and / or the presence of dead cells.

[0228] Organoids The present invention provides organoid or epithelial stem cell population, which can be obtained or obtained by the method of the present invention.Thus, in some embodiments, said method further comprises obtaining and / or isolating organoid.The organoid or epithelial stem cell population of the present invention comprises breast cells.Thus, breast organoid is provided, which comprises a population of breast epithelial stem cells.Preferably, the organoid or epithelial stem cell population of the present invention is composed of breast cells.

[0229] A method for growing breast cells is currently described in Pasic et al. (2011) Genes & Development 25: 1641-1653, but this culture method and the resulting cell structure are different from the culture method and the resulting cell structure provided by the present invention. The method described in Pasic et al. uses a culture system that includes (i) amphiregulin or EGF (HER1 ligand) and (ii) FGF2 or FGF7. The method described in Pasic et al. does not use a culture medium that includes a combination of ErbB3 / 4 ligand and Lgr5 agonist or FGFR2b ligand as used in the present invention. Pasic et al. tested a culture medium that includes NRG1-β1, an ErbB3 / 4 ligand, but did not find that breast organoids can grow in this culture medium. Pasic et al. did not test a culture medium that includes a combination of ErbB3 / 4 ligand and Lgr5 agonist or FGFR2b ligand. Using the culture system of Pasic et al., a cell structure with a morphology different from that of the organoid of the present invention is formed. In particular, the cell structures of Pasic et al. have a budding morphology, which has ragged edges, whereas the organoids obtained using the methods of the present invention lack this morphology, and instead have smooth cyst-like structures or morphologies resembling a bunch of grapes. It has been observed that cultures using the Pasic et al. system stop growing after 2-3 passages (see Example 4 herein). In contrast, the present inventors have discovered that, in some embodiments, using the methods of the present invention, it is possible to grow mammary epithelial stem cells for more than 14 passages.

[0230] A method for culturing circulating tumor cells is described in Yu et al. (2014) Science 345(6193):216-220. However, the authors of this paper did not provide instructions for culturing breast epithelial stem cells obtained from normal breast tissue, primary tumors, or metastases. Furthermore, this culture system is different from the present invention and results in a different structure compared to the organoids of the present invention. In particular, Yu uses a culture method that includes EGF and FGF2. Yu et al. do not use a culture medium that includes a combination of ErbB3 / 4 ligand and Lgr5 agonist or FGFR2b ligand as used in the present invention. Indeed, Yu et al. do not use any culture medium that includes ErbB3 / 4 ligand. Using Yu's method, aggregates of single tumor cells are formed without strong cell-cell adhesion. Continuous epithelium is not formed using this method. In contrast, organoids obtained using the method of the present invention show cell-cell adhesion and continuous epithelium. In some embodiments, cell-cell adhesion is strong.

[0231] Thus, the present invention provides breast organoids with cyst-like structures. In some embodiments, the cyst-like structures include a central lumen. In some embodiments, the central lumen of the cyst-like structures does not include any cells. In other embodiments, the central lumen of the cyst-like structures includes breast epithelial stem cells, and optionally, the central lumen is filled with breast epithelial stem cells. Furthermore, the present invention provides breast organoids with a morphology similar to a bunch of grapes. Furthermore, the present invention provides breast epithelial stem cells, breast epithelial stem cell populations, or breast organoids that have been cultured or can be cultured for more than three passages. Furthermore, the present invention provides a culture medium that includes a combination of ErbB3 / 4 ligand and Lgr5 agonist and / or FGFR2b ligand.

[0232] The organoids of the invention, obtainable by proliferation of stem cells, preferably provide a cell population that resembles its in vivo counterpart.

[0233] In some embodiments, the organoid comprises at least one epithelial stem cell. The term "stem cell" preferably refers to a cell that can divide and generate additional epithelial stem cells or give rise to differentiated progeny, for example, progenitor cells that can divide and further differentiate into one or more cell types. However, in some embodiments, the terms "stem cell" and "progenitor cell" may be used synonymously. In some cases, progenitor cells may dedifferentiate into stem cells, and thus, when progenitor cells are cultured in the culture medium of the present invention, progenitor cells may dedifferentiate into stem cells. For example, it should be understood that in preferred organoids, the majority of cells are proliferating cells (i.e., dividing cells) that retain undifferentiated phenotype. Although some spontaneous differentiation may occur, the cell population is generally a proliferating population. The epithelial stem cells used in the present invention are preferably pluripotent, oligopotent, or unipotent organ-specific adult stem cells. Stem cells are not totipotent stem cells. In some embodiments, stem cells are not pluripotent stem cells. Said organoids also have characteristic structures resulting from these cellular characteristics, as detailed below.

[0234] Image analysis can be used to evaluate the characteristics of cells in culture, such as cell morphology; cell structure; evidence of apoptosis or cell lysis; and the composition and structure of organoid.Many types of imaging analysis are well known in the art, such as electron microscope, confocal microscope, stereo microscope, fluorescence microscope.Histological analysis can reveal basic structure and cell type.

[0235] The organoid of the present invention preferably has a three-dimensional structure. That is, the organoid is preferably a three-dimensional organoid. In a preferred embodiment, the organoid only contains epithelial cells. That is, the organoid is devoid of non-epithelial cells. Because the culture medium of the present invention is specifically designed to grow epithelial stem cells, for example, Lgr5+ epithelial stem cells. Therefore, even if other cell types are temporarily present in the culture medium, for example, in the tissue fragment that is the starting material of the present invention, these cells may not survive, but instead are replaced by the long-term growth of stem cells, which produces a pure epithelial cell population.

[0236] In some embodiments, epithelial cells surround lumen.In some embodiments, epithelial cells surround lumen are polarized (meaning that proteins are expressed differently on the apical or basolateral side of epithelial cells).In some embodiments, said organoid comprises stem cells that can actively divide and preferably can differentiate into all major differentiated cell lineages present in corresponding in vivo tissue.

[0237] In some embodiments, the organoid of the present invention has a region that is formed by multiple layers.In some embodiments, the organoid of the present invention has a single layer region that appears to be formed by multiple layers, which is referred to herein as " pseudostratified" cell region.In some embodiments, the single layer of pseudostratified cells folds over, so that it encloses a lumen between the folds.

[0238] In some embodiments, the organoid of the present invention comprises a single monolayer that folds (or invaginates) to form two or more layers.Sometimes, it is difficult to distinguish between the folded (or invaginated) monolayer and the layered cell region.In some embodiments, the organoid comprises both the layered cell region and the folded monolayer region.In some embodiments, the lung organoid of the present invention has a region that is formed by multiple layers and a region that comprises a single cell monolayer.In some embodiments, the organoid of the present invention comprises a single cell monolayer or is composed of a single cell monolayer.

[0239] In some embodiments, the organoid of the present invention comprises epithelial cells or consists of epithelial cells.In some embodiments, said organoid comprises epithelial cell monolayer or consists of epithelial cell monolayer.In some embodiments, organoid does not have non-epithelial cells.

[0240] Illustrative examples of organoids made according to the present invention are shown in the attached drawings.In one embodiment, the organoids of the present invention have the structure essentially as shown in Figure 2 (for example, in some embodiments, normal organoids have the structure essentially as shown in Figure 2B for W894N or R1100N organoid strains; or cancer organoids have the structure essentially as shown in Figure 2A for W1007T, W1012T or W859T organoid strains) or as shown in Figure 6.In one embodiment, the organoids of the present invention show cell staining essentially as shown in Figure 5 or Figure 6.

[0241] In some embodiments, said organoid is mainly cystic structure, and there is little budding structure.In some embodiments, the organoid made according to the present invention does not have budding structure.Cystic structure mainly comprises a single layer, but some stratified cell regions may exist.

[0242] "Cystic" means that the organoids are roughly spherical. "Budded" means that the organoids have multiple regions growing out from a basic structure.

[0243] Under the improved culture conditions of the present invention, mammary organoids exhibited cystic organoid structures rather than the budding structures seen under previous culture conditions.

[0244] The present invention also provides an organoid, which is a three-dimensional organoid comprising epithelial cells surrounding a central lumen, preferably an organoid obtainable by the method of the present invention.

[0245] In some embodiments, the organoids comprise a monolayer of cells. In other embodiments, the organoids comprise a multi-layered epithelium.

[0246] In some embodiments, the organoids are derived from normal breast tissue and have a cystic structure. In some embodiments, the normal organoids comprise a single layer of basal and luminal cells surrounding a central lumen.

[0247] In some embodiments, organoid is obtained from cancerous breast tissue and has cystic structure.In some embodiments, said organoid (i) comprises a central lumen, a number of small lumens or does not comprise a lumen, and / or (ii) comprises an epithelial cell monolayer or multi-layered epithelium.Thus, in some embodiments, breast cancer organoid of the present invention comprises an epithelial cell monolayer surrounding a lumen.In other embodiments, breast cancer organoid comprises multi-layered epithelium and a number of small lumens.In further embodiments, breast cancer organoid comprises multi-layered epithelium and does not comprise a lumen.Breast cancer organoid optionally comprises solid spheres of tumor cells.

[0248] The tumor status of cancer cells or cancer organoids can be confirmed by any suitable method. For example, karyotyping of cells or organoids can be performed to evaluate whether aneuploidy exists. If aneuploidy exists, the cells or organoids will be suggested to be tumor cells or tumor organoids. Tumor cells or cancer organoids may also be identified by adding p53 stabilizers to culture medium. Tumor cells and cancer organoids usually contain p53 mutations that result in p53 stabilizers that do not affect p53 degradation. Thus, unlike normal cells, tumor cells can often avoid senescence induced by adding p53 stabilizers (e.g., Nutlin-3) to culture medium.

[0249] In some embodiments, breast cancer organoids are positively stained for luminal cell markers, such as keratin-8 (Krt-8), and negatively stained for basal cell markers, such as keratin-14 (Krt-14) or keratin-5 (Krt-5).Thus, in some embodiments, breast cancer organoids contain luminal cells but do not contain basal cells.In some embodiments, breast cancer organoids express KRT-8 at a level comparable to that found in normal breast tissue or non-cancerous breast organoids.In some embodiments, breast cancer organoids express KRT-5 and / or KRT-14 at a lower level compared to that found in normal breast tissue or non-cancerous breast organoids.

[0250] The breast cancer organoid of the present invention may express a lower level of CDH1 compared to that found in normal breast tissue or non-cancerous breast organoid.The epithelial-mesenchymal transition marker (e.g., VIM, ZEB1) may be highly expressed in breast cancer organoid compared to normal breast tissue or non-cancerous breast organoid.The breast cancer organoid may show a lower level of E-cadherin expression compared to normal breast tissue or non-cancerous breast organoid.

[0251] The breast cancer organoids of the present invention may express other breast cancer cell markers, such as epithelial membrane antigen, cancer antigen 15-3 (CA15-3), cancer antigen 27.29 (CA27.29), carcinoembryonic antigen (CEA), urokinase plasminogen activator (uPA), plasminogen activator inhibitor (PAI-1), podocalyxin, EZH2, and / or kallikrein-10.

[0252] The present invention provides an organoid or epithelial stem cell population that has been cultured in the culture medium of the present invention for at least 2 months, for example, at least 10 weeks, at least 12 weeks, at least 14 weeks, at least 16 weeks, at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least 1 year.Preferably, the cells are human cells.Preferably, the cells are passaged about every 1-4 weeks.

[0253] Also provided are organoids or epithelial stem cell populations that have been or can be passaged for more than 3 passages, more than 4 passages, more than 5 passages, more than 6 passages, more than 7 passages, more than 8 passages, more than 9 passages, more than 10 passages, more than 11 passages, more than 12 passages, more than 13 passages, or more than 14 passages.

[0254] Advantageously, the use of ErbB3 / 4 ligand allows breast cells and organoids to be cultured for a long time.It is not possible to culture and grow human breast epithelial stem cells or breast organoids for more than 2 months in the culture medium described in Pasic et al., WO2012 / 014076, or WO2012 / 168930, where they are passaged at least three times.Therefore, such breast organoids or breast epithelial stem cell populations that are cultured for a long time did not exist before the present invention.Therefore, organoids or stem cell populations that are cultured / passaged or can be cultured / passaged as described herein are provided.

[0255] In the context of the present invention, tissue fragment is a part of adult tissue, preferably human adult tissue, for example human breast.Tissue may be normal (healthy) tissue, diseased tissue or infected tissue.Therefore, preferably, the organoid specified herein is not a tissue fragment.Organoid is preferably obtained using cells derived from adult tissue, preferably epithelial stem cells derived from adult tissue, optionally epithelial stem cells derived from adult tissue fragment.In some embodiments, organoid is obtained using epithelial stem cells derived from living tissue or living tissue fragment that express Lgr5.Therefore, in the context of the present invention, in some embodiments, tissue fragment comprises Lgr5+ stem cells.

[0256] In one embodiment, the organoid is cultured using the method of the present invention (preferably using the culture medium of the present invention), and thus is in contact with extracellular matrix. Preferably, the organoid is embedded in a non-mesenchymal extracellular matrix. In the context of the present invention, "in contact" means physical or mechanical or chemical contact. Physical or mechanical or chemical contact means that force must be used to separate the organoid from the extracellular matrix. In some embodiments, the extracellular matrix is ​​a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, such as Matrigel (BD Biosciences) or Cultrex® Basement Membrane Extract (Trevigen, Inc.). Thus, the culture medium of the present invention may optionally further comprise the cell population of the present invention and / or one or more organoids of the present invention.

[0257] The present invention provides a composition comprising an organoid or cell of the present invention, an extracellular matrix, and a culture medium, preferably a culture medium of the present invention.

[0258] Uses of Organoids There is an interest in culture media and methods for culturing stem cells to form, maintain and grow organoids, such as breast organoids. Organoids contain stem cells, such as epithelial stem cells, that retain undifferentiated phenotype and self-renewal properties, but also have differentiating progeny that can grow into tissue-like structures. As with populations of related or identical cells, breast organoids closely mimic the basic physiology of breast tissue and can be used in toxicity or drug assays. Organoids may also be useful for culturing pathogens for which there is currently no suitable tissue culture or animal model. Furthermore, such organoids may be useful in regenerative medicine, for example, in post-radiation and / or post-surgery breast repair.

[0259] It is clear that stem cells and their differentiated progeny have many clinical and research applications. For all these applications, reproducible culture methods to prepare sufficient numbers of cells of appropriate quality are paramount. For example, for effective drug screening, conditions must be carefully controlled and rigorous culture methods are required to control cell differentiation and proliferation, thus generating pure populations of phenotypically and karyotypically identical cells. Similarly, for cell-based therapies, where cultured cells may be provided directly to patients, the cells must be genetically and phenotypically correct to avoid undesirable immune responses or cell fates when provided to the patient.

[0260] The present invention provides the use of the organoid or the expanded cell population of the present invention for use in medicine.Therefore, the present invention provides the use of the organoid or the expanded cell population of the present invention for use in the treatment of disorders, conditions or diseases.The present invention provides the use of the organoid or the expanded cell population of the present invention for use in drug screening, (drug) target validation, (drug) target discovery, toxicology and toxicity screening, personalized medicine, regenerative medicine, and / or as ex vivo cell / organ model, for example, disease model.

[0261] The cell and organoid cultured according to the medium and method of the present invention is believed to represent the in vivo situation faithfully.This applies to the cell population and organoid that grows and proliferates from normal tissue, and the cell population and organoid that grows and proliferates from diseased tissue.Therefore, the organoid or proliferated cell population of the present invention can not only provide normal ex vivo cell / organ model, but can also be used as ex vivo disease model.

[0262] The organoid 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 animal hosts.Therefore, the organoid of the present invention can be used as disease model that represents infectious conditions.In some embodiments of the present invention, the organoid can be used in the development and / or production of vaccines.

[0263] Thus, diseases that can be studied by the organoids of the present invention include, for example, genetic, metabolic, pathogenic, inflammatory diseases, and the like, including, but not limited to, breast cancer, adenomas, ductal ectasia, fibrocystic changes (e.g., adenomas, dysplasias, hyperplasias, ductal lesions, lobular lesions, papillomas), mastitis, mammary syphilis, mammary tuberculosis, and mammary actinomycosis.

[0264] Traditionally, cell lines, and more recently iPS cells, have been used as ex vivo cell / organ and / or disease models (see, for example, Robinton et al. Nature 481, 295, 2012). However, these methods have many problems and drawbacks. For example, cell lines cannot be obtained from all patients (only certain biopsies result in successful cell lines). Thus, cell lines cannot be used in personalized diagnostics and medicine. iPS cells usually require some degree of genetic engineering to reprogram the cells to a specific cell fate. Alternatively, iPS cells are subjected to culture conditions that affect karyotypic integrity, and therefore culture time must be minimized (this also applies to human embryonic stem cells). This means that iPS cells cannot accurately represent the in vivo situation, but instead are an attempt to mimic the behavior of in vivo cells. There is also genetic instability in cell lines and iPS cells.

[0265] In contrast, the organoid of the present invention provides a genetically stable platform that faithfully represents the in vivo situation.The organoid of the present invention can also be continuously propagated to be a good source of genetically stable cells.In particular, the proliferating population can be "split"."Split" means that the organoid is divided and all the cells of the organoid are separated and placed in a new culture dish or flask.The separated cells are taken out of the organoid and then can be cultured and propagated themselves to generate new organoids containing further propagated populations, and then the new organoids can be split again.Splitting is also referred to as "passage" in this specification. The organoids of the present invention may be cultured for one or more passages, for example, 1st passage, 2nd passage, 3rd passage, 4th passage, 5th passage, 6th passage, 7th passage, 8th passage, 9th passage, 10th passage, 15th passage, 20th passage, 25th passage, 30th passage, or more, for example, 20-30th passage, 30-35th passage, 32-40th passage, or more. In some embodiments, the growing cell population or organoids are split every 3 days to 10 weeks, every 1-10 weeks, every 1-4 weeks, or every 10 days to 3 weeks. In some embodiments, the organoids are passaged about every 10 days to 3 weeks. Thus, the organoids of the present invention can provide a sustained source of genetically stable cell material. Thus, the organoids of the present invention can be used to gain mechanistic insight into various diseases and therapeutic agents, to perform in vitro drug screens, to evaluate potential therapeutic agents, to identify possible targets (e.g., proteins) for future development of novel (drug) therapies, and / or to explore gene repair in the context of cell replacement therapy.

[0266] For these reasons, the organoids or expanded cell populations of the present invention can be tools for drug screening, target validation, target discovery, toxicology and toxicity screening, and personalized medicine.

[0267] Drug Screening Preferably, for high throughput purposes, the expanded stem cell population or organoid of the present invention is cultured in a multi-well plate, for example, a 96-well plate or a 384-well plate. Molecular libraries are used to identify molecules that affect the organoid. Preferred libraries include antibody fragment libraries, peptide phage display libraries, peptide libraries (e.g., LOPAP™, Sigma Aldrich), lipid libraries (BioMol), synthetic compound libraries (e.g., LOP AC™, Sigma Aldrich), or natural compound libraries (Specs, TimTec). In addition, gene libraries can be used to induce or suppress the expression of one or more genes in the progeny of stem cells. These gene libraries include cDNA libraries, antisense libraries, and siRNA libraries or other non-coding RNA libraries. Cells are preferably exposed to multiple concentrations of test agents for a certain period of time. At the end of the exposure period, the culture is evaluated. The term "affect" is used to cover any change in cells, including but not limited to proliferation, morphological changes, and reduction or elimination of cell death. Said expanded stem cell population or organoid of the present invention can also be used to identify the drug that specifically targets breast cancer cell, breast cancer cell or breast cancer organoid of the present invention, but does not target normal breast cell, for example, the normal expanded stem cell population or normal organoid of the present invention.Of course, drug screening does not always need to be high-throughput, and the organoid and cell of the present invention are also useful for testing individual drug candidates.

[0268] Organoids are also useful for a wide range of drug discovery purposes.It will be understood by those skilled in the art that the organoids of the present invention can be widely applied as drug screening tools for infectious, inflammatory and neoplastic conditions of human breast and other diseases of breast.In some embodiments, the organoids of the present invention may be used for cancer drug screening.

[0269] In some embodiments, the expanded cell population, for example, the organoid of the present invention or the organoid obtained using the medium and method of the present invention, can be used to test the suitability of a library of chemicals, antibodies, natural products (plant extracts), etc., or a specific known drug for use as a drug, cosmetic, and / or prophylactic. For example, in some embodiments, cell biopsy material from a patient of interest, for example, tumor cells from a cancer patient, can be cultured using the culture medium and method of the present invention, and then treated with a compound or compound library of interest or one or more drugs. It can then be ascertained which compound effectively modifies, kills, and / or treats the patient's cells. This allows the responsiveness of a particular patient to a particular drug to be tested, and thus allows treatment to be tailored to a particular patient. This therefore allows for a personalized medicine approach. An additional advantage of using organoids to identify drugs in this way is that normal organoids (organoids derived from healthy tissue) can also be screened to see which drugs and compounds have minimal effects on healthy tissue. This allows the screening of drugs with minimal off-target activity or unwanted side effects.

[0270] In this way, any number of disease drugs can be screened.For example, the organoid of the present invention can be used to screen drugs for breast cancer, adenoma, ductal ectasia, fibrocystic changes (e.g., adenoma, dysplasia, hyperplasia, ductal lesion, lobular lesion, papilloma), mastitis, mammary syphilis, mammary tuberculosis, and mammary actinomycosis.

[0271] Test parameters are determined by the disease of interest.For example, when screening cancer drugs, cancer cell death is usually the ultimate goal.In other embodiments, metabolism or gene expression can be evaluated to study the effect of screening compounds and drugs on cells or organoids of interest.

[0272] Thus, the present invention provides a method for screening therapeutic or prophylactic drugs, comprising the steps of: Culturing the expanded cell population (e.g., organoid) of the present invention, e.g., with a culture medium of the present invention; exposing the expanded cell population of the invention (e.g., organoids) to one or a library of candidate molecules; assessing the expanded cell population (e.g., organoids) for any effects, e.g., any cellular changes, e.g., reduced or absent proliferation, morphological changes, and / or cell death; Identifying candidate molecules that cause said effect as potential drugs.

[0273] In some embodiments, computer or robotic assisted culture and data collection methods are used to increase the throughput of screening.

[0274] In some embodiments, the expanded cell population (e.g., organoid) is obtained from a patient biopsy. In some embodiments, the candidate molecule that induces a desired effect in the cultured expanded cell population (e.g., organoid) is administered to the patient.

[0275] Thus, in one aspect, a method for treating a patient is provided, comprising the steps of: (a) obtaining a biopsy from a diseased tissue of interest in a patient; (b) screening for a suitable drug using the screening method of the present invention, for example, culturing the expanded cell population of the present invention (e.g., organoids) with the culture medium of the present invention; exposing the expanded cell population (e.g., organoids) of the invention to one or a library of known drugs; Evaluating the expanded cell population (e.g., organoids) for any effects, e.g., any cellular changes, e.g., reduced or absent proliferation, morphological changes, and / or cell death; Identifying candidate molecules that cause said effect as suitable drugs; and (c) treating said patient with the medicament obtained in step (b).

[0276] For example, the present invention provides a method of treating breast cancer in a patient, comprising testing the patient's responsiveness to one or more drugs of interest using the drug screening methods of the present invention, and then treating the patient with the drug if the patient is found to respond to the drug.

[0277] Similarly, there is provided a breast cancer drug for use in treating a breast cancer patient, said treatment comprising testing the patient's responsiveness to a drug using the drug screening method of the invention, and if the patient is found to respond to the drug, treating the patient with the drug.

[0278] Examples of drugs for use in the present invention that can be tested using the personalized medicine approach outlined above include Abitrexate (methotrexate), Abraxane (paclitaxel albumin stabilized nanoparticle formulation), Ado-Trastuzumab Emtansine, Adriamycin PFS (doxorubicin hydrochloride), Adriamycin RDF (doxorubicin hydrochloride), Adrucil (fluorouracil), Afinitor (everolimus), Anastrozole, Aredia (pamidronate disodium), Arimidex (anastrozole), Aromasin (exemestane), Capecitabine (C apecitabine, Clafen (cyclophosphamide), cyclophosphamide, Cytoxan (cyclophosphamide), docetaxel, doxorubicin hydrochloride, Efudex (fluorouracil), Ellence (epirubicin hydrochloride), epirubicin hydrochloride, everolimus, exemestane, Fareston (toremifene), Faslodex (Fulvestrant), Femara (letrozole), Fluoroplex (fluorouracil), Fluorouracil, Folex (methotrexate), Folex PFS (methotrexate), Fulvestrant, Gemcitabine hydrochloride, Gemzar (gemcitabine hydrochloride), Goserelin acetate, Herceptin (trastuzumab), Ixabepilone, Ig Ixempra (ixabepilone), Kadcyla (adotrastuzumab emtansine), lapatinib ditosylate, letrozole, Megace (megestrol acetate), megestrol acetate, methotrexate, methotrexate LPF (methotrexate), Mexate (methotrexate), Mexate-AQ (methotrexate), Neosar (cyclophosphamide),Nolvadex (tamoxifen citrate), Novaldex (tamoxifen citrate), paclitaxel, paclitaxel albumin-stabilized nanoparticle formulation, pamidronate disodium, Perjeta (pertuzumab), pertuzumab, tamoxifen citrate, Taxol (paclitaxel), Taxotere (docetaxel), trastuzumab, toremifene, Tykerb (lapatinib dicitrate), Xeloda (capecitabine), and Zoladex (goserelin acetate).

[0279] Drugs for use in the present invention that may be tested using a personalized medicine approach may be tested alone or in combination with one or more other drugs.

[0280] For example, (a) doxorubicin hydrochloride, Adriamycin PFS (doxorubicin hydrochloride), or Adriamycin RDF (doxorubicin hydrochloride) may be tested in combination with (b) cyclophosphamide, Clafen (cyclophosphamide), Cytoxan (cyclophosphamide), or Neosar (cyclophosphamide), optionally further combined with (c)(i) paclitaxel, paclitaxel albumin stabilized nanoparticle formulation, Abraxane (paclitaxel albumin stabilized nanoparticle formulation), or Taxol (paclitaxel), (c)(ii) fluorouracil, Adolcil (fluorouracil), Efudex (fluorouracil), or Fluoroplex (fluorouracil), or (c)(iii) docetaxel or Taxotere (docetaxel). Thus, drug combinations of AC, AC-T, CAF, and TAC may be tested using a personalized medicine approach according to the present invention.

[0281] For example, (a) cyclophosphamide, Clafen (cyclophosphamide), Cytoxan (cyclophosphamide), or Neosar (cyclophosphamide) may be tested in combination with (b) fluorouracil, Adolsil (fluorouracil), Efudex (fluorouracil), or Fluoroplex (fluorouracil), and (c)(i) methotrexate, avitrexate (methotrexate), Folex (methotrexate), Folex PFS (methotrexate), methotrexate LPF (methotrexate), mexate (methotrexate), or mexate-AQ (methotrexate), or (c)(ii) epirubicin hydrochloride or ellence (epirubicin hydrochloride). Thus, drug combinations of CMF and FEC may be tested using the personalized medicine approach according to the present invention.

[0282] In some embodiments, the drug is used to treat, prevent, or ameliorate conditions such as genetic, metabolic, pathogenic, inflammatory diseases, including, but not limited to, breast cancer, adenomas, ductal dilatation, fibrocystic changes (e.g., adenomas, metaplasias, hyperplasias, ductal lesions, lobular lesions, papillomas), mastitis, breast syphilis, breast tuberculosis, and breast actinomycosis.

[0283] Target Discovery In some embodiments, the organoid of the present invention or the cells grown using the culture medium and method of the present invention can be used for target discovery.The cells of organoids generated from healthy or diseased tissue can be used for target identification.The organoid of the present invention can be used for the drug target discovery of breast cancer, adenoma, ductal dilatation, fibrocystic changes (e.g., adenoma, metaplasia, dysplasia, hyperplasia, ductal lesion, lobular lesion, papilloma), mastitis, breast syphilis, breast tuberculosis, and breast actinomycosis.

[0284] The cells and organoids cultured according to the medium and method of the present invention are considered to represent the in vivo situation faithfully.For this reason, the cells and organoids cultured according to the medium and method of the present invention can be a tool for discovering new (molecular) targets in certain diseases.

[0285] To search for new drug targets, a compound (e.g., siRNA) library can be used to transduce cells and inactivate specific genes. In some embodiments, siRNA is transduced into cells to inhibit the function of a (large) group of genes. Any functional readout of a group of genes or a specific cellular function can be used to confirm whether the target is relevant to the study. Disease-specific readouts can be confirmed using assays known in the art. For example, cell proliferation is assayed to test genes involved in cancer. For example, the Topflash assay described herein can be used to detect changes in Wnt activity caused by siRNA inhibition. If reduced growth or cell death occurs, the corresponding siRNA-associated genes can be identified by methods known in the art. These genes are potential targets for inhibiting the growth of these cells. Once identified, the specificity of the identified targets for the tested cellular process will need to be confirmed by methods known in the art. These methods can be used to identify new molecules as potential drug targets for therapy.

[0286] Target and drug validation screening Patient-specific organoids obtained from diseased and / or normal tissues can be used for target validation of molecules identified in high-throughput screening. The same is true for validation of compounds identified as potential therapeutics in high-throughput screening. It may be useful to use primary patient material grown in organoid culture system to test for false positives from high-throughput drug discovery cell line studies, etc.

[0287] In some embodiments, the expanded stem cell populations (e.g., organoids of the present invention) can be used for validation of compounds identified as potential drugs or cosmetics in high throughput screens.

[0288] Toxicity assays Moreover, instead of using cell line in toxicity assay of potential new drugs or known drugs, the above-mentioned expanded stem cell population (for example, the organoid of the present invention) can be used.Preferably, these embodiments use normal cells or normal organoids.However, it is also envisioned that cancer cells or cancer organoids may be used.

[0289] Toxicity screens function similarly to drug screens (above), but test the toxic effect of a drug, not its therapeutic effect. Thus, in some embodiments, the effect of the candidate compound is a toxic effect.

[0290] Pathogen culture Additionally, the expanded stem cell populations (e.g., organoids of the present invention) can be used to culture pathogens, such as mouse mammary tumor virus (MMTV), human mammary tumor virus (HMTV), Epstein-Barr virus (EBV), high-risk human papillomavirus (HPV), Mycobacterium tuberculosis, Streptococcus uberis, Streptococcus dysgalactiae, Escherichia coli, Staphylococcus aureus, or Streptococcus agalactiae.

[0291] Therapeutic monitoring and identification of causes of drug resistance Furthermore, the above-mentioned expanded stem cell population (for example, the organoid of the present invention) can be used to monitor the progression of disease in patients and to monitor the progression of disease in expanded stem cell population.Thus, for example, to identify the cause of drug resistance and the appropriate therapy, expanded breast cancer stem cell population can be obtained from breast cancer patients, and then this patient can be treated with a certain drug or drug combination, and if the patient develops resistance to the drug or drug combination, a second expanded breast cancer stem cell population can be obtained from the patient and compared with the first breast cancer stem cell population.

[0292] Thus, the above-mentioned expanded stem cell population (for example, the organoid of the present invention) can be used to identify the cause of drug resistance.Therefore, for example, to identify the cause of drug resistance, expanded breast cancer stem cell population can be obtained from breast cancer patient, and then this patient can be treated with a certain drug or drug combination, and if the patient develops resistance to the drug or drug combination, a second expanded breast cancer stem cell population can be obtained from the patient and compared with the first breast cancer stem cell population.

[0293] By identifying genetic changes in resistant stem cell populations compared to stem cell populations that respond to drug treatment, the causes of drug resistance may be identified.

[0294] Regenerative Medicine and Transplantation The culture containing the expanded stem cell population (for example, the organoid of the present invention) is useful in regenerative medicine, for example, in breast tissue repair after radiation and / or surgery, for example, after mastectomy, or in tissue repair of damaged breast tissue.For example, the breast epithelial stem cells with BRCA mutation can be obtained from cancer patients, and then BRCA mutation can be repaired, thereby generating autologous breast epithelial stem cells, which can then be transplanted back into the cancer patients.

[0295] In another embodiment, the expanded epithelial stem cells are reprogrammed to take on the relevant tissue fate. Moreover, it will be clear to those skilled in the art that gene therapy can be used in methods aimed at repairing damaged or diseased tissue. For example, adenoviral or retroviral gene delivery vehicles can be used to deliver genetic information, such as DNA and / or RNA, to stem cells. Those skilled in the art can replace or repair specific genes targeted in gene therapy. For example, normal genes can be inserted into non-specific locations in the genome to replace non-functional genes. In another example, abnormal gene sequences can be substituted for normal gene sequences by homologous recombination. Or selective reversion can return genes to normal function. A further example is the change in the regulation (the degree to which genes are turned on or off) of specific genes. Preferably, stem cells are treated ex vivo by gene therapy approaches and then introduced into a mammal, preferably a human, in need of treatment.

[0296] Since small biopsies taken from adult donors can be grown without obvious limitations or genetic harm, said technology may be useful for making transplantable epithelium for regenerative purposes.Furthermore, in some embodiments, organoids embedded in ECM or in contact with ECM can be transplanted into mammals, preferably humans.In another embodiment, organoids and ECM can be transplanted into mammals, preferably humans, simultaneously.

[0297] Those skilled in the art will understand that ECM can be used as a 3D scaffold to obtain tissue-like structures, including the expanded cell population or organoids according to the present invention. Such structures can then be implanted into patients by methods well known in the art. ECM scaffolds can be synthetically made using ECM proteins such as collagen and / or laminin. Alternatively, ECM scaffolds can be obtained by "decellularising" an isolated organ or tissue fragment, leaving a scaffold consisting of ECM (see, for example, Macchiarini et al. The Lancet, Volume 372, Issue 9655, Pages 2023-2030, 2008). In some embodiments, ECM scaffolds can be obtained by decellularising an organ or tissue fragment, optionally said organ or tissue fragment originating from the breast.

[0298] As mentioned above, the present invention provides an organoid or cell population of the present invention for use in transplantation into a mammal, preferably a human.

[0299] Advantageously, using the present invention, small biopsies can be taken from adult donors and grown without apparent restriction or genetic harm. Thus, the techniques provided herein may be useful for generating transplantable epithelia for regenerative purposes.

[0300] The patient is preferably a human, but may alternatively be a non-human mammal, such as a cat, dog, horse, cow, pig, sheep, rabbit, or mouse.

[0301] Thus, within the scope of the present invention is a method of treating a human or non-human animal patient with cell therapy. Such cell therapy includes applying the stem cells or organoids of the present invention to the patient by any suitable means. In particular, such treatment involves the regeneration of damaged tissue. According to the present invention, the patient can be treated with allogeneic or autologous stem cells or organoids. An "autologous" cell is a cell that originates from the same organism as the organism into which the cell is reintroduced for cell therapy, for example to regenerate tissue. However, the cell is not necessarily isolated from the same tissue as the tissue into which the cell is introduced. Autologous cells do not require patient matching to overcome rejection problems. An "allogeneic" cell is a cell that originates from an individual of the same species, but different from the individual into which the cell is reintroduced for cell therapy, for example to regenerate tissue. To prevent rejection problems, some degree of patient matching may still be required.

[0302] Generally, the cells or organoids of the present invention are introduced into the patient's body by injection or transplantation.Generally, the cells are directly injected into the tissue that the cells are intended to act on.The syringe containing the cells of the present invention and a pharmacologic acceptable carrier is included within the scope of the present invention.The catheter attached to the syringe containing the cells of the present invention and a pharmacologic acceptable carrier is included within the scope of the present invention.

[0303] A person skilled in the art will be able to select the appropriate method and route of administration depending on the material being transplanted (i.e., cell population, single cells in cell suspension, organoids, or organoid fragments).

[0304] As mentioned above, the cells of the present invention can be used in tissue regeneration.To achieve this function, the cells can be directly injected or transplanted into damaged tissue, where they can grow and eventually differentiate into the cell type required.Alternatively, organoids can be directly injected or transplanted into damaged tissue.Breast tissues that are amenable to treatment include all damaged tissues, particularly those that may be damaged by disease (e.g., cancer), injury, trauma, autoimmune reaction, or viral or bacterial infection.In some embodiments of the present invention, the cells or organoids of the present invention are used to regenerate breast.

[0305] For example, in one embodiment, the cells or organoids of the present invention are injected into patients using a Hamilton syringe.Accordingly, the present invention provides a syringe that contains the cells or organoids of the present invention.

[0306] A person skilled in the art will know what the appropriate dosage of the cells or organoids of the present invention is for the particular condition to be treated.

[0307] In one embodiment, the cells or organoids of the present invention are administered in a dissolved state into microspheres or microparticles of various compositions into an artery and irrigated into the tissue or part of the damaged organ that needs regeneration. Generally, such administration is performed using a catheter. The catheter may be one of a wide variety of balloon catheters used for angioplasty and / or cell delivery, or a catheter designed for the specific purpose of delivering cells to a specific localized area of ​​the body. In certain applications, the cells or organoids may be encapsulated in microspheres with a diameter of about 15 μm, made of a number of different biodegradable compounds. This method may allow the cells or organoids administered intravascularly to remain at the site of injury and not enter the systemic circulation through the capillary network on the first pass. Retention on the arterial side of the capillary network may also facilitate migration to the extravascular space.

[0308] In another embodiment, the cells or organoids of the present invention may be transplanted into damaged tissue while attached to a biocompatible graft. In this embodiment, the cells may be attached to a biocompatible graft in vitro and then transplanted into a patient. As will be clear to those skilled in the art, any one of a number of adhesives may be used to attach the cells to the graft prior to transplantation. By way of example only, such adhesives may include fibrin, one or more members of the integrin family, one or more members of the cadherin family, one or more members of the selectin family, one or more cell adhesion molecules (CAMs), one or more members of the immunoglobulin family, and one or more artificial adhesives. This list is given by way of example only and is not intended to be limiting. It will be clear to those skilled in the art that any combination of one or more adhesives may be used.

[0309] In another embodiment, the cell or organoid of the present invention can be embedded in the matrix before the matrix is ​​implanted in the patient.Generally, the matrix is ​​implanted in the damaged tissue of the patient.Examples of the matrix include collagen-based matrix, fibrin-based matrix, laminin-based matrix, fibronectin-based matrix and artificial matrix.This list is only given as an example and is not intended to be limiting.

[0310] In further embodiments, the cells or organoids of the present invention may be implanted or injected into a patient together with matrix-forming components.This may allow the cells to form a matrix after injection or implantation, and ensure that the cells or organoids remain in the appropriate position in the patient.Examples of matrix-forming components include fibrin glue, liquid alkyl, cyanoacrylate monomers, plasticizers, polysaccharides such as dextran, ethylene oxide-containing oligomers, block copolymers such as poloxamer and Pluronic, non-ionic surfactants such as Tween and Triton "8", and artificial matrix-forming components.This list is only given by way of example and is not intended to be limiting.It will be clear to those skilled in the art that any combination of one or more matrix-forming components can be used.

[0311] In a further embodiment, the cells or organoids of the present invention may be contained within a microsphere. Within this embodiment, the cells may be encapsulated in the center of the microsphere. Also within this embodiment, the cells may be embedded in the matrix material of the microsphere. The matrix material may comprise any suitable biodegradable polymer, including but not limited to alginate, polyethylene glycol (PLGA), and polyurethane. This list is given by way of example only and is not intended to be limiting.

[0312] In further embodiments, the cells or organoids of the present invention may be attached to a medical device for transplantation. Examples of such medical devices include stitches and artificial skin. This list is given by way of example only and is not intended to be limiting. It will be clear to those skilled in the art that cells may be attached to a medical device by various methods. For example, cells or organoids may be attached to a medical device using fibrin, one or more members of the integrin family, one or more members of the cadherin family, one or more members of the selectin family, one or more cell adhesion molecules (CAMs), one or more of the immunoglobulin family, and one or more artificial adhesives. This list is given by way of example only and is not intended to be limiting. It will be clear to those skilled in the art that any combination of one or more adhesives may be used.

[0313] Other Uses of the Organoids of the Invention The cells or organoids of the present invention may be used to define patient groups for drugs. The cells or organoids of the present invention may also be used to pre-screen patients in phase I or phase II trials. For example, the cells or organoids of the present invention may be used to identify patients who will undergo clinical trials. The cells or organoids of the present invention may also be used in diagnosis after a drug is approved. For example, the cells or organoids of the present invention may be used to identify patients who are most likely to benefit from a particular drug, to identify patients who may be at high risk of experiencing serious side effects as a result of treatment with a particular drug, or to monitor the response to treatment with a particular drug in order to adjust the treatment to achieve improved safety or efficacy.

[0314] The cells or organoids of the present invention may in some embodiments be used in veterinary therapy, for example, the cells or organoids may be used to treat bovine mastitis.

[0315] Compositions and other forms of the present invention The present invention provides pharmaceutical compositions comprising the cells or organoids of the present invention and further comprising a pharma- ceutically acceptable carrier or excipient.

[0316] The present invention provides a composition comprising the culture medium of the present invention and stem cells.The present invention also provides a composition comprising the culture medium of the present invention and organoid.Furthermore, the present invention provides a composition comprising the culture medium of the present invention and extracellular matrix.

[0317] The present invention also provides a composition comprising the culture medium of the present invention, the extracellular matrix, and the stem cells of the present invention.The present invention also provides a composition comprising the culture medium of the present invention, the extracellular matrix, and one or more organoids of the present invention.The present invention also provides a culture medium supplement that can be used to generate the culture medium disclosed herein.A "culture medium supplement" is a mixture of components that cannot support stem cells by themselves, but when combined with other cell culture components, enables or improves stem cell culture.Therefore, the supplement can be used to generate the functional cell culture medium of the present invention by combining with other cell culture components to generate a suitable medium formulation.The use of culture medium supplements is well known in the art.

[0318] The present invention provides a culture medium supplement comprising the ErbB3 / 4 ligand according to the present invention.The supplement may contain any ErbB3 / 4 ligand (or a combination of ErbB3 / 4 ligand) disclosed herein.The supplement may also contain one or more additional cell culture components disclosed herein, such as one or more cell culture components selected from the group consisting of amino acids, vitamins, inorganic salts, carbon energy sources, and buffer solutions.

[0319] The culture medium or culture medium supplement may be a concentrated liquid culture medium or supplement (e.g., 2x to 250x concentrated liquid culture medium or supplement) or a dried culture medium or supplement. Both liquid culture medium or supplement and dried culture medium or supplement are well known in the art. The culture medium or supplement may be freeze-dried.

[0320] The culture medium or culture medium supplement of the present invention is typically sterilized, for example, by ultraviolet light, heating, irradiation, or filtration, before use to prevent contamination. The culture medium or culture medium supplement may be frozen (e.g., at -20°C or -80°C) for storage or transportation. In some embodiments, the culture medium may be stored as a liquid (e.g., at about 4°C). In some embodiments, the culture medium may be split and stored as two components: a frozen component (e.g., at about -20°C to about -80°C) and a liquid component (e.g., at about 4°C). In particular, it is preferred that temperature-sensitive or time-sensitive degradable materials are included in the frozen component, while less sensitive materials (e.g., DMEM or FCS) are stored in liquid form and thus may be included in the liquid component for storage and shipping.

[0321] The present invention also provides a hermetically sealed container that also contains the culture medium or culture medium supplement of the present invention.Hermetically sealed container may be preferred for transporting or storing the culture medium or culture medium supplement disclosed herein to prevent contamination.The container may be any suitable container, such as flask, plate, bottle, jar, vial or bag.

[0322] The present invention also provides a kit that comprises the culture medium, culture medium supplement, and / or composition of the present invention.In some embodiments, the kit further comprises at least one other additional component, for example at least one other additional component selected from the list that comprises ECM (for example, Matrigel (trademark) or Cultrex (registered trademark) Basement Membrane Extract), cell population, and organoid.

[0323] General Provisions The term "comprising" encompasses "including" as well as "consisting." For example, a composition "comprising" X may consist of only X, or it may include something more, e.g., X+Y.

[0324] The word "substantially" does not exclude "completely", e.g. a composition that is "substantially free" of Y may be completely free of Y. Where necessary, the word "substantially" may be omitted from the definition of the invention.

[0325] The term "about" in relation to a numerical value x is arbitrary and means, for example, x±10%.

[0326] Unless otherwise specified, a process that includes mixing two or more components does not require a particular order of mixing. Thus, the components can be mixed in any order. If there are three components, two components can be combined with each other, and then this combination can be combined with a third component, etc.

[0327] Various aspects and embodiments of the invention are explained in further detail below by way of example, it being understood that modifications of detail can be made without departing from the scope of the invention. EXAMPLES

[0328] Example 1 - Tissue processing and mammary organoid culture Summary 20 breast tumors were sampled, of which 3 were triple negative (15%, Fig. 1A and Fig. 13). We successfully established 3 tumor organoid lines (split ratio >1:2, passage >7). Eight additional cultures are currently being expanded, and in our experience, they are almost certain to be successful organoid lines (Fig. 2 and Fig. 13). It is unclear that a particular subtype was selected (Fig. 1B).

[0329] So far, we have established three promising cultures from the 19 normal samples obtained (Figure 2, Figure 13).

[0330] Culture conditions The first 12 samples were used to optimize cell isolation and organoid culture conditions. Samples 13–20 were grown in six different conditions.

[0331] Changing parameters for cell isolation To isolate viable cells, we cultured the cells with several digestive enzymes (collagenase, dispase, trypsin) at different concentrations (1–5 mg / ml) in different media (AdDF+++, basal medium, basal F) with or without stirring. 7 / 10 N medium) for different periods (1-2 h, 12 h). The composition of these basal media is shown in Figure 14. The optimal protocol is summarized below.

[0332] Parameter changes for organoid culture To allow organoid growth, we tested several culture conditions (6-12 conditions / sample) simultaneously. Based on previous experience working with other human tissues, we tested several growth factors in addition to our basal medium (Figure 14). Other parameters tested were BME (basement membrane extract) density (50-100%) replacing Matrigel, the use of tissue culture treated plates vs. suspension plates, and 2% O. 2 For surroundings O 2An example of the effect of different culture conditions is shown for normal and tumor organoid lines obtained from patient W855 (Figure 3).

[0333] Protocol for tissue processing and mammary organoid culture Upon arrival, photograph the tissue and slice it at 1-3 mm 3 The tissue was cut into 100x100mm pieces. Two random pieces were snap frozen and stored at -80°C for DNA isolation. Two random pieces were fixed in formalin for histopathological analysis and immunohistochemistry, and the remainder was processed for viable cell isolation. The remaining tissue was minced, washed with 10 ml of AdDF+++, and diluted with basic F containing 1-2 mg / ml collagenase (Sigma-C9407). 7 / 10 The tissue fragments were digested in 10 ml of N medium at 120 rpm and 37°C for 1-2 hours. The digested tissue suspension was successively sheared using 10 ml and 5 ml plastic pipettes and a flamed glass Pasteur pipette. After each shearing step, the suspension was filtered through a 100 μm filter. The retained tissue fragments entered the next shearing step with approximately 10 ml of AdDF+++. 2% FCS was added to the strained suspension before centrifugation at 400g. The pellet was resuspended in 10 ml of AdDF+++ and centrifuged again at 400. In case of a visible red pellet, the red blood cells were lysed with 2 ml of red blood cell lysis buffer (Roche-11814389001) for 5 min at room temperature, followed by the addition of 10 ml of AdDF+++ and centrifugation at 400g. The pellet was resuspended in cold 10mg / ml Cultrex growth factor reduced BME type 2 (Trevigen-3533-010-02) and a 40μl drop of the BME cell suspension was allowed to solidify on a pre-warmed 24-well suspension culture plate (Greiner-M9312) for 10-20 minutes at 37°C. Once gelation was complete, 400μl of organoid medium (basal F 7 / 10 N, basic F 7 / 10 EN or Basic F 7 / 10 PN) was added to each well and the plate was incubated with 2% O 2 Or ambient O 2Either humidified 37℃ / 5% CO 2 Transferred to an incubator.

[0334] The medium was changed every 4 days and organoids were passaged every 1–4 weeks. Cystic organoids were resuspended in 2 ml of cold AdDF+++ and mechanically sheared through a flame-prepared glass Pasteur pipette. High-density organoids were resuspended in 2 ml of TrypLE Express (Invitrogen-12605036), incubated at room temperature for 1–5 min, and dissociated by mechanically shearing through a flame-prepared glass Pasteur pipette. After addition of 10 ml of AdDF+++ and centrifugation at 300 g or 400 g, respectively, organoid fragments were resuspended in cold BME and reseeded at a ratio (1:1–1:6) that allowed the formation of new organoids. Single cell suspensions were initially seeded at high density and reseeded at low density approximately 1 week later.

[0335] Example 2 - Characterization of established breast tumor organoid lines Breast tumor organoid lines W854T, W855T, and W859T are derived from the base F 7 / 10 They grow readily in N medium. They were therefore characterized in more detail.

[0336] The status of these tumors was confirmed by karyotyping and revealed variable degrees of aneuploidy (Figure 4).

[0337] Furthermore, strain W854T grew readily in medium containing 5 μM Nutlin-3, indicating the presence of mutant p53. RNA and DNA were isolated and sent to BI for gene expression profiling and exome sequencing.

[0338] Histological and immunofluorescence analyses confirmed that ER, PR, and HER2 profiles were preserved between the original tumors and established tumor organoid lines even after seven passages (Figures 5 and 6).

[0339] Whereas the normal organoid line, W855N, consists of mutually exclusive basal and luminal cells, the tumor organoids express only the luminal cell marker keratin-8 (Figures 6 and 7). E-cadherin expression is present in the tumor but is less abundant compared to the normal organoid line, indicating an epithelial origin (Figures 6 and 7). However, epithelial-mesenchymal transition (EMT) markers are mildly upregulated in the tumor organoid lines, W855T and W859T, which is consistent with the inconsistent phenotype of the tumor organoid lines, W855T and W859T (Figures 6 and 7).

[0340] Example 3 - Low-Throughput Drug Screening To test the feasibility of breast tumor organoids for drug screening, lines W854T, W855T, and W859T were plated in duplicate in a 96-well format and exposed to medium containing the EGFR inhibitor Iressa (1 nM-30 μM in 1:3 dilution steps, S1025 Selleck Chemicals) or the p53 stabilizer Nutlin-3 (1 nM-30 μM in 1:3 dilution steps, 10004372 Cayman Chemicals) for 7 days. Photographs of organoids were taken every 3-4 days (Figure 8) and cell viability was measured on day 7 using the CellTiter-Glo 2.0 assay (G9241, Promega) (Figure 9).

[0341] All three organoid lines respond to Iressa at a concentration of about 10 μM, indicating a dependency on EGFR signaling. Nutlin-3 does not inhibit growth up to a concentration of 30 μM, possibly with the exception of W855T, indicating the presence of mutant p53 (Figure 9). Breast tumor organoids are suitable for low-throughput drug screening based on cell survival in a 96-well format. We are currently testing this assay in a 384-well plate format that has been successfully established for colon cancer organoids.

[0342] Example 4 - Culture medium of Pasic et al. does not support long-term growth Normal breast organoids in Pasic culture medium slowed down their proliferation at passage 2 and stopped at passage 3. 7 / 10 Organoids from the same patient grown in medium basal E continued to proliferate up to passage 4 (see FIG. 10). Normal breast organoids from a different patient were grown in medium basal F. 7 / 10 Proliferate in EN to at least passage 6.

Claims

1. A method for obtaining or culturing a cancer organoid, comprising culturing a cancer epithelial stem cell or a cancer organoid consisting of a cancer epithelial stem cell in a culture medium containing a p53 stabilizer, wherein the p53 stabilizer enhances the cellular concentration of p53 and stimulates p53-dependent expression, and the cancer epithelial stem cell comprises a p53 mutation.

2. A culture medium for obtaining or culturing a cancer organoid, comprising a p53 stabilizer, wherein the p53 stabilizer enhances the cellular concentration of p53 and stimulates p53-dependent expression, wherein the cancer organoid is composed of cancer epithelial stem cells, and the cancer epithelial stem cells comprise a p53 mutation.

3. Use of a p53 stabilizer in a method for obtaining or culturing a cancer organoid, wherein the p53 stabilizer enhances a cellular concentration of p53 and stimulates p53-dependent expression, the cancer organoid is composed of cancer epithelial stem cells, and the cancer epithelial stem cells contain a p53 mutation.

4. 4. The method of claim 1 or the use of claim 3, wherein the p53 stabilizer is a member of the Nutlin family.

5. 5. The method or use according to claim 4, wherein the p53 stabilizer is a member of the Nutlin family and is selected from Nutlin-1, Nutlin-2 or Nutlin-3.

6. 6. The method or use of claim 5, wherein the p53 stabilizer is Nutlin-3.

7. A method or use according to claim 1 or any one of claims 3 to 6, wherein the cancer epithelial stem cells or cancer organoids are derived from the lung, small intestine, stomach, pancreas, liver, prostate, and breast.

8. The culture medium of claim 2, wherein the p53 stabilizer is a member of the Nutlin family.

9. The culture medium of claim 8, wherein the p53 stabilizer is a member of the Nutlin family and is selected from Nutlin-1, Nutlin-2 or Nutlin-3.

10. 10. The culture medium of claim 9, wherein the p53 stabilizer is Nutlin-3.

11. 11. The culture medium of claim 2, further comprising one or more receptor tyrosine kinase ligands and a BMP inhibitor.

12. 11. The culture medium of claim 2, further comprising one or more of a receptor tyrosine kinase ligand, a BMP inhibitor, and a TGF-β inhibitor.

13. 13. The culture medium of claim 12, wherein the TGF-β inhibitor is A83-01.

14. (a) the receptor tyrosine kinase ligand is selected from EGF, one or more FGFR2b ligands, and / or HGF; and / or (b) the BMP inhibitor is Noggin; 14. A culture medium according to claim 12 or claim 13.

15. The culture medium according to claim 14, wherein the receptor tyrosine kinase ligand is one or more FGFR2b ligands selected from FGF7 and / or FGF10.

16. (a) p38 MAP kinase inhibitor; (b) Gastrin; (c) nicotinamide; (d) a Rock inhibitor; (e) B27; (f) N-acetylcysteine; (g) a Notch inhibitor; (h) Prostaglandin pathway activators 16. The culture medium of any one of claims 2 and 8 to 15, further comprising one or more components selected from:

17. (i) p53 stabilizers, Lgr5 agonists, BMP inhibitors, B27, N-acetylcysteine, nicotinamide, ROCK inhibitors, TGF-β inhibitors, p38 MAP kinase, FGF7, and FGF10; or (ii) a p53 stabilizer, one or more receptor tyrosine kinase ligands, a BMP inhibitor and a TGF-β inhibitor, a p38 inhibitor, and / or a ROCK inhibitor; or (iii) p53 stabilizers, one or more receptor tyrosine kinase ligands, BMP inhibitors and gastrin, nicotinamide, Notch inhibitors, and / or prostaglandin pathway activators.

16. The culture medium according to any one of claims 2 and 8 to 15, comprising:

18. (i) the p38 MAP kinase inhibitor is SB202190; and / or (ii) the ROCK inhibitor is Y-27632; and / or (iii) the Notch inhibitor is DAPT and / or DBZ; and / or (iv) the prostaglandin pathway activator is PGE2 and / or AA; and / or (v) the p53 stabilizer is Nutlin-3; and / or (vi) the TGF inhibitor is A83-01; and / or (vii) the BMP inhibitor is Noggin; A culture medium according to any one of claims 16 to 17.

19. (i) one or more additional receptor tyrosine kinase ligands; and / or (ii) one or more Wnt agonists The culture medium of any one of claims 2 and 8 to 18, further comprising one or more additional components selected from:

20. 20. The culture medium of claim 19, wherein the one or more additional receptor tyrosine kinase ligands are selected from EGF, HGF, amphiregulin, TGF-α, and / or PDGF.

21. The culture medium of claim 19, wherein the one or more Wnt agonists are one or more Lgr5 agonists.

22. The culture medium of claim 21 , wherein the one or more Lgr5 agonists are Rspondin.

23. 20. The culture medium of claim 19, wherein the one or more Wnt agonists is Wnt 3a.

24. 24. The culture medium of any one of claims 2 and 8 to 23, further comprising a cAMP pathway activator and / or a BMP pathway activator.

25. 25. The culture medium of claim 24, wherein the cAMP pathway activator is forskolin and / or the BMP pathway activator is BMP7.

26. The method according to any one of claims 1 and 4 to 7, wherein the culture medium is a culture medium according to any one of claims 2 and 8 to 25.

27. ​​A p53 mutant tumor organoid in a culture medium described in any one of claims 2 and 8 to 25.

28. Use of a p53 mutant tumor organoid as defined in claim 27 in drug discovery screening; toxicity assays; investigating histogenesis, cell lineages, or differentiation pathways; studying gene expression, including recombinant gene expression; investigating mechanisms involved in tissue damage or repair; investigating inflammatory and infectious diseases; studying mechanisms of pathogenesis; or studying mechanisms of cell transformation or the etiology of cancer.

29. Use of the p53 mutant tumor organoid of claim 27 in the manufacture of a medicament for the treatment of disorders, conditions or diseases requiring regenerative medicine.

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