Method for producing organoid and organoid

Abstract

Provided is a technique for producing an organoid that is larger in size and more functionally excellent than conventional organoids. The present invention employs a method for producing an organoid, the method comprising: a step (A1) for culturing an organoid, in which an organoid produced from undifferentiated cells, mesenchymal cells, and vascular cells that differentiate into a first organ is brought into a secretion factor secreted from a second organ; and a step (A2) for culturing the organoid cultured in the step (A1). In the step (A1) and the step (A2), the dissolved oxygen partial pressure of a medium in contact with the organoid is 20% or less. The dissolved oxygen partial pressure of the medium in contact with the organoid in the step (A1) is lower than the dissolved oxygen partial pressure of the medium in contact with the organoid in the step (A2).

Description

Method for producing organoids and organoids

[0001] The present invention relates to a method for producing organoids and organoids. This application claims priority based on Japanese Patent Application No. 2024-140043, filed on August 21, 2024, the contents of which are incorporated herein by reference.

[0002] Toward the creation of human organs, technology for producing organoids using iPS cells is being developed. Organoids are immature structures that can differentiate into organs. For example, Patent Document 1 and Non-Patent Document 1 describe the production of undifferentiated cells that differentiate into liver cells, mesenchymal cells, and vascular cells from iPS cells, and the production of organoids from these cells.

[0003] Patent No. 7233717

[0004] Takebe et al., Massive and Reproducible Production of Liver Buds Entirely from Human Pluripotent Stem Cells, Cell Rep. 2017 21, 2661-2670

[0005] However, the organoids described in Patent Document 1 and Non-Patent Document 1 are smaller in size and less functional than human organs. Therefore, an objective of the present invention is to provide a technology for producing organoids that are larger in size and more functionally superior than conventional organoids.

[0006] The present invention includes the following aspects: [1] A method for producing organoids, comprising: a step (A1) of contacting organoids prepared from undifferentiated cells, mesenchymal cells, and vascular cells that differentiate into a first organ with secretory factors secreted from a second organ and culturing the organoids; and a step (A2) of culturing the organoids cultured in the step (A1), wherein the dissolved oxygen partial pressure of the medium in contact with the organoids in the step (A1) and the dissolved oxygen partial pressure of the medium in contact with the organoids in the step (A2) are 20% or less of 100 kPa (100%), and the dissolved oxygen partial pressure of the medium in contact with the organoids in the step (A1) is lower than the dissolved oxygen partial pressure of the medium in contact with the organoids in the step (A2). [2] The method for producing organoids according to [1], wherein the first organ is the liver. [3] The method for producing organoids according to [1], wherein the second organ is a placenta. [4] The method for producing organoids according to [2], wherein the secreted factor is IL1α. [5] A method for producing organoids, comprising a step of contacting IL1α with organoids produced from undifferentiated cells that differentiate into liver, mesenchymal cells, and vascular cells, and culturing the organoids. [6] An organoid produced by the method for producing organoids according to any one of [1] to [5]. [7] An organoid in which the proportion of cells that are HNF4α-positive and Ki67-positive is 1% or more relative to the number of HNF4α-positive cells (100%).

[0007] According to the present invention, a technique for producing organoids that are larger in size and have better functionality than conventional organoids can be provided.

[0008] 1 shows the results of pimonidazole staining for livers at E9.5 to E11.5 in Experimental Example 1. FIG. 2 shows a graph indicating the percentage of pimonidazole-positive areas for livers at E9.5 to E11.5 in Experimental Example 1. FIG. 3 shows the results of observing liver progenitor cells (KO-HE) under a fluorescent microscope in Experimental Example 2. FIG. 4 shows the results of measuring the area of ​​KO-HE in Experimental Example 2. FIG. 5 shows the results of measuring the volume of organoids in Experimental Example 2. FIG. 6 shows a photograph of an example of an obtained colony in Experimental Example 3. FIG. 7 shows a graph indicating the number of small colonies and large colonies in Experimental Example 3. FIG. 8 shows the results of measuring the number of small colonies and large colonies in Experimental Example 3. FIG. 9 shows the results of measuring the number of small colonies and large colonies in Experimental Example 3. ＋ CK19 ＋ 3 is a photograph of cells expressing hepatic progenitor cells (HNF4α). FIG. 4 is a photograph of organoids obtained in Experimental Example 4. FIG. 5 is a graph showing the results of measuring the area of ​​organoids obtained in Experimental Example 4. FIG. 6 is a photograph of organoids obtained in Experimental Example 5. FIG. 7 is a graph showing the ratio of the area of ​​hepatic progenitor cell regions in organoids after late culture to the area of ​​hepatic progenitor cell regions in organoids after early culture in Experimental Example 5. FIG. 8 is a diagram showing the scheme of Experimental Example 6. FIG. 9 is a photograph of organoids obtained in Experimental Example 6, after immunostaining with Ki67 and HNF4α. ＋ ) relative to the number of proliferating liver progenitor cells (Ki67 ＋ HNF4α ＋ 6. It is a graph showing the expression level of a marker for hepatic progenitor cells for organoids in Experimental Example 6. It is a graph showing the results of measuring the volume of organoids in Experimental Example 6. It is a graph showing the secretion amount of human albumin of organoids in Experimental Example 6. It is a graph showing the expression level of a marker for hepatic cells for organoids in Experimental Example 6.

[0009] (Organoid Culture Method: First Aspect) The organoid production method according to the first aspect includes the following steps (A1) and (A2).

[0010] Step (A1): A step of contacting the organoid with a secretory factor secreted from a second organ and culturing it.

[0011] Step (A2): A step of culturing the organoids cultured in step (A1).

[0012] In step (A1), the dissolved oxygen partial pressure of the medium contacting the organoids, and in step (A2), the dissolved oxygen partial pressure of the medium contacting the organoids, is 20% or less relative to 100 kPa (100%). In step (A1), the dissolved oxygen partial pressure of the medium contacting the organoids is lower than the dissolved oxygen partial pressure of the medium contacting the organoids in step (A2). In this specification, the culture conditions in step (A1) may be referred to as "hypoxic conditions". In addition, the culture conditions in step (A2) may be referred to as "hypoxic conditions".

[0013] In this specification, the oxygen partial pressure (%) in the atmosphere containing the culture medium and the dissolved oxygen partial pressure (%) in the culture medium refer to the ratio relative to a total pressure of 100 kPa (100%).

[0014] <Step (A1)> In step (A1), the organoid is cultured by contacting it with a secretory factor secreted from a second organ.

[0015] <Organoid> As used herein, the term "organoid" refers to a structure that can differentiate into an organ upon maturation.

[0016] The organ species that organoid can differentiate into is preferably mammalian.Mammalian may be human or non-human animal.Non-human animal may be, for example, pet, livestock etc., more specifically, for example, pig, cow, horse, dog, cat, monkey etc.

[0017] In the first embodiment, the organoid to be cultured is prepared from undifferentiated cells that differentiate into a first organ, mesenchymal cells, and vascular cells.

[0018] <<First organ>> In the first aspect, the organ that organoid differentiates to form is called the first organ. This organoid may be referred to as the "organoid of the first organ". Examples of the first organ include ectodermal organs such as the brain, spinal cord, adrenal medulla, epidermis, hair, nails, skin glands, sensory organs, peripheral nerves, and lens; mesodermal organs such as the spleen, kidneys, ureters, heart, blood, gonads, adrenal cortex, muscle, skeleton, dermis, connective tissue, and mesodermal organs such as the mesoderm; and endodermal organs such as the liver, pancreas, digestive tract (pharynx, esophagus, stomach, intestinal tract), lung, thyroid gland, parathyroid gland, urinary tract, and thymus. Among these, the liver, pancreas, and intestinal tract are preferred, and the liver is more preferred.

[0019] In this specification, the organoid that can differentiate into a certain organ may be referred to as the organoid of that organ.Specifically, for example, the organoid that can differentiate into liver may be referred to as liver organoid, and the organoid that can differentiate into pancreas may be referred to as pancreatic organoid.

[0020] Among the terms used by those skilled in the art, for example, liver bud, liver diverticula, liver organoid, pancreatic (dorsal or ventral) buds, pancreatic diverticula, pancreatic organoid, intestinal bud, intestinal diverticula, and intestinal organoid are included in the term "organoid" in this specification.

[0021] Whether a structure is an organoid or not can be confirmed, for example, by analyzing the expression of proteins that serve as organ markers in the structure. Examples of organ markers include markers known to those skilled in the art.

[0022] For example, liver organoid markers include HHEX, SOX2, HNF4A, AFP, ALB, etc.; pancreatic organoid markers include PDX1, SOX17, SOX9, etc.; intestinal organoid markers include CDX2, SOX9, etc.

[0023] In a first embodiment, the organoid is prepared from undifferentiated cells, mesenchymal cells, and vascular cells that differentiate into a first organ. These cells will be described in detail below.

[0024] [Undifferentiated cells that differentiate into a first organ] As used herein, "undifferentiated cells that differentiate into a first organ" refers to undifferentiated cells that differentiate to form a first organ and exhibit the function of the first organ. The undifferentiated cells include stem cells of the first organ, progenitor cells of the first organ, and endodermal cells.

[0025] Among the terms used by those skilled in the art, for example, hepatoblast, hepatic progenitor cells, pancreatoblast, hepatic precursor cells, pancreatoblast, pancreatic progenitors, pancreatic progenitor cells, pancreatic precursor cells, endocrine precursors, intestinal progenitor cells, intestinal precursor cells, intermediate mesoderm, and metanephric mesenchymal precursor are included. Cells, multipotent nephron progenitor cells, renal progenitor cells, cardiac mesoderm, cardiovascular progenitor cells, cardiac progenitor cells, and the like are encompassed by the "undifferentiated cells that differentiate into the first organ."

[0026] The undifferentiated cells that differentiate into the first organ may be cells prepared from pluripotent stem cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells) according to known methods.

[0027] Undifferentiated cells that differentiate into liver cells can be produced from iPS cells by, for example, conventional methods (Takebe et al., Massive and Reproducible Production of Liver Buds Entirely from Human Pluripotent Stem Cells, Cell Rep. 2017 21, 2661-2670; Supplemental Information).

[0028] Specific methods for producing undifferentiated cells that differentiate into liver cells from iPS cells include the following. First, iPS cells are cultured in the presence of 10 μM ROCK Inhibitor Y-27632 and 1 mM sodium butyrate using a dish coated with Laminin 511 E8 fragment (e.g., Nippi, iMatrix-511). Next, the cells are cultured using RPMI-1640 containing 1% B27 supplement (Thermo Fisher Scientific), 100 ng / ml activin A, and 50 ng / ml Wnt3a. Next, by culturing the cells using RPMI-1640 containing 1% B27, 10 ng / ml human basic FGF, and 20 ng / ml human BMP4, undifferentiated cells that differentiate into liver cells can be produced.

[0029] Undifferentiated cells that differentiate into kidneys can be produced from iPS cells by, for example, conventional methods (Shi M et al., Human ureteric bud organoids recapitulate branching morphogenesis and differentiate into functional collecting duct cell types, Nat Biotechnol. 2023 Feb;41(2):252-261.). Specifically, iPS cells are first cultured in the presence of Activin A, BMP4, a GSK3 inhibitor, and FGF2. The cultured cells are then cultured in the presence of a TGF-β inhibitor, FGF2, a BMP inhibitor, and retinoic acid. The cultured cells are then cultured in the presence of GDNF and retinoic acid. These steps allow the production of cells that differentiate into kidneys.

[0030] Undifferentiated cells that differentiate into the intestinal tract can be produced from iPS cells by, for example, a conventional method (Spence JR et al., Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro, Nature. 2011 Feb 3; 470(7332): 105-109.). Specifically, iPS cells are cultured in the presence of Activin A. Then, by culturing the cells in the presence of FGF4 and Wnt3a, cells that differentiate into the hindgut can be produced.

[0031] Undifferentiated cells that differentiate into brain cells can be generated from iPS cells, for example, by conventional methods (Mayhew CN and Singhania R., A review of protocols for brain organoids and applications for disease modeling, STAR Protoc. 2023 Mar 17; 4(1): 101860.).

[0032] Whether a cell is an undifferentiated cell that will differentiate into a first organ can be confirmed by examining the expression of a marker protein. For example, markers for undifferentiated cells that will differentiate into the liver include HHEX, SOX2, HNF4A, AFP, ALB, etc.; markers for undifferentiated cells that will differentiate into the pancreas include PDX1, SOX17, SOX9, etc.; and markers for undifferentiated cells that will differentiate into the intestine include CDX2, SOX9, etc.

[0033] [Mesenchymal cells] As used herein, the term "mesenchymal cells" encompasses cells that have differentiated into connective tissue cells, which exist in connective tissue derived from the mesoderm and form a support structure for cells that function in tissues, as well as undifferentiated cells that can differentiate into connective tissue cells. As mesenchymal cells, undifferentiated cells that can differentiate into connective tissue cells are preferred.

[0034] Whether a cell is an undifferentiated cell capable of differentiating into a connective tissue cell can be confirmed by analyzing the expression of a marker protein, etc. Examples of the marker protein include Stro-1, CD29, CD44, CD73, CD90, CD105, CD133, CD271, and Nestin.

[0035] Among terms used by those skilled in the art, for example, septum mesenchyme, septum transversum mesenchyme, mesenchymal stem cells, mesenchymal progenitor cells, and mesenchymal cells are encompassed in the mesenchymal cells of this specification.

[0036] The mesenchymal cells may be cells prepared from pluripotent stem cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells) according to known methods.

[0037] Mesenchymal cells can be produced from iPS cells, for example, by conventional methods (Takebe et al., Massive and Reproducible Production of Liver Buds Entirely from Human Pluripotent Stem Cells, Cell Rep. 2017 21, 2661-2670; Supplemental Information).

[0038] Specific methods for producing mesenchymal cells from iPS cells include the following: First, iPS cells are cultured in the presence of Laminin 511 E8 fragment and 10 μM ROCK Inhibitor Y-27632. Next, the medium is replaced with mesoderm induction medium (a 1:1 mixture of DMEM and F12 supplemented with 1% Glutamax, 1% B27, 8 μM CHIR99021, and 25 ng / ml BMP4), followed by the addition of 2 ng / ml activin A and 10 ng / ml PDGFBB for culture. Next, the medium is replaced with mesenchymal cell induction medium (StemPro-34 SFM medium supplemented with 10 ng / ml FGF2 and 10 ng / ml PDGFBB) for culture.

[0039] [Vascular cells] As used herein, "vascular cells" refers to cells that have differentiated into cells that constitute blood vessels, or undifferentiated cells that can differentiate into cells that constitute blood vessels. Vascular cells are preferably undifferentiated cells that can differentiate into cells that constitute blood vessels. The undifferentiated cells include vascular stem cells, vascular progenitor cells, and mesodermal cells.

[0040] Examples of vascular cells include vascular endothelial cells, vascular endothelial precursor cells, endocardial precursor cells, and hemangioblasts, with vascular endothelial cells being preferred.

[0041] Whether a certain cell is a vascular endothelial cell or not can be confirmed by analyzing the expression of a marker protein, etc. Examples of the marker protein include TIE2, VEGFR-1, VEGFR-2, VEGFR-3, CD41, etc.

[0042] Among terms used by those skilled in the art, for example, endothelial cells, umbilical vein endothelial cells, endothelial progenitor cells, endothelial precursor cells, vasculogenic progenitors, and hemangioblasts are included in the vascular cells of this specification.

[0043] Vascular cells may be cells prepared from pluripotent stem cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells) according to known methods.

[0044] Vascular cells can be produced from iPS cells, for example, by conventional methods (Takebe et al., Massive and Reproducible Production of Liver Buds Entirely from Human Pluripotent Stem Cells, Cell Rep. 2017 21, 2661-2670; Supplemental Information).

[0045] Specific methods for producing vascular cells from iPS cells include the following. First, iPS cells are cultured in the presence of Laminin 511 E8 fragment and 10 μM ROCK Inhibitor Y-27632. Next, the medium is replaced with priming medium (a 1:1 mixture of DMEM and F12 supplemented with 1% Glutamax, 1% B27, 8 μM CHIR99021, and 25 ng / ml BMP4) and cultured. Next, the priming medium is replaced with endothelial cell induction medium (StemPro-34 SFM medium supplemented with 200 ng / ml VEGF and 2 μM forskolin) and cultured. Next, endothelial cells localized with CD144 and CD31 are selected by FACS analysis. Then, the cells are cultured in an endothelial cell growth medium (StemPro-34 SFM medium supplemented with 50 ng / ml VEGF-A) on a fibronectin-coated dish.

[0046] In the first embodiment, the organoid to be cultured is prepared from undifferentiated cells, mesenchymal cells, and vascular cells that differentiate into a first organ. Specifically, the organoid is prepared by mixing undifferentiated cells, mesenchymal cells, and vascular cells that differentiate into a first organ in a medium and culturing them. Organoids can be prepared, for example, by conventional methods (Takebe et al., Massive and Reproducible Production of Liver Buds Entirely from Human Pluripotent Stem Cells, Cell Rep. 2017 21, 2661-2670; Supplemental Information).

[0047] The ratio of the numbers of undifferentiated cells that differentiate into the first organ, mesenchymal cells, and vascular cells to be mixed is, for example, 2 to 1 mesenchymal cells and 10 to 5 vascular cells per 10 undifferentiated cells that differentiate into the first organ.

[0048] As a medium for producing organoids, for example, endothelial cell growth medium (EGM), hepatocyte culture medium (HCM), a medium supplemented with dexamethasone, oncostatin M, and hepatocyte growth factor may be used. In the medium, the concentration of dexamethasone is preferably 1 nM to 1 μM, more preferably 10 nM to 1000 nM, and even more preferably 20 nM to 500 nM. In the medium, the concentration of oncostatin M is preferably 0.1 to 1000 ng / mL, more preferably 1 to 200 ng / mL, and even more preferably 5 to 100 ng / mL. In the medium, the concentration of the hepatocyte growth factor is preferably 0.1 to 1000 ng / mL, more preferably 1 to 200 ng / mL, and even more preferably 5 to 100 ng / mL.

[0049] <<Second Organ>> The second organ is an organ different from the first organ. Examples of the second organ include the placenta, kidney, liver, heart, lung, spleen, esophagus, stomach, thyroid gland, parathyroid gland, thymus, gonads, brain, and spinal cord, with the placenta being preferred.

[0050] <<Secreted Factor>> The secreted factor secreted from the second organ is contained in the culture medium for culturing the organoid described below.As the secreted factor secreted from the second organ, a mimic of the secreted factor secreted from the second organ may be used.The secreted factor secreted from the second organ may include, for example, cytokines, growth factors, etc. Examples of cytokines include interleukin-1α (IL1α), interleukin-1β, interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-8, interleukin-10, interleukin-11, interleukin-12, interleukin-13, interleukin-14, interleukin-15, interleukin-16, interleukin-17, interleukin-18, interferon α, interferon β, interferon γ, granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), monocyte chemotactic protein-1 (MCP-1), erythropoietin (EPO), thrombopoietin (TPO), and Flk-2 / Flt-3 ligand (FL).

[0051] Examples of cell growth factors and cell differentiation factors include vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), osteonectin, angiopoietin, hepatocyte growth factor (HGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), nerve growth factor (NGF), leukemia inhibitory factor (LIF), stem cell growth factor (SCF), bone morphogenetic protein (BMP), interferon-α, interferon-β, interferon-γ, tumor necrosis factor-α, tumor necrosis factor-β, and the like.

[0052] Of the above secretory factors, IL1α is preferred.

[0053] "Contacting the organoid with a secretory factor secreted from a second organ" may mean that the medium in which the organoid is cultured contains the secretory factor secreted from the second organ.

[0054] The concentration of the secretory factor secreted from the second organ in the culture medium for culturing organoids is not limited as long as the effects of the present invention are achieved, and can be appropriately determined by a person skilled in the art. When the secretory factor secreted from the second organ is IL1α, the concentration of IL1α in the culture medium is preferably 0.1 to 1000 ng / mL, more preferably 1 to 200 ng / mL, and even more preferably 2 to 100 ng / mL.

[0055] The origin of the secretory factor to be contacted with the organoid is not particularly limited, and secretory factors derived from various organisms can be used. Among these, secretory factors derived from mammals are preferred. Examples of mammals include humans, mice, rats, cows, pigs, and rabbits. It is more preferred that the animal species from which the secretory factor to be contacted with the organoid is derived is the animal species from which the cells forming the organoid are derived. The nucleotide sequence of the mRNA encoding the secretory factor or the amino acid sequence of the protein can be obtained from sequence databases such as GenBank. For example, mature human IL1α consists of the amino acid sequence set forth in SEQ ID NO: 1.

[0056] Methods for producing a secretory factor include methods using cells that express the secretory factor. The origin of the secretory factor-expressing cells (such as biological species and culture form) is not particularly limited, and they may be cells that stably express the secretory factor, or cells that transiently express the secretory factor.

[0057] Secretory factor-expressing cells can be produced using known gene recombination techniques. That is, secretory factor-expressing cells can be produced by inserting DNA encoding the desired secretory factor into a known expression vector and introducing the resulting expression vector into an appropriate host cell. The nucleotide sequence of the gene encoding the desired secretory factor can be obtained from known databases such as GenBank.

[0058] The secretory factor expressed by the secretory factor-expressing cell may be a fragment of the secretory factor, or may contain an amino acid sequence other than the amino acid sequence of the secretory factor, as long as it has its activity. The amino acid sequence other than the amino acid sequence of the secretory factor is not particularly limited, and examples include the amino acid sequence of an affinity tag. The secretory factor may be a processed mature form, etc., as long as it has its activity. Furthermore, the amino acid sequence of the secretory factor does not need to be completely identical to an amino acid sequence obtainable from a publicly known database such as GenBank, and may be substantially the same as an amino acid sequence obtainable from a publicly known database, as long as it has the activity of the secretory factor.

[0059] Examples of amino acid sequences that are substantially identical to the amino acid sequences of secretory factors obtainable from publicly known databases include amino acid sequences obtained by deleting, inserting, substituting, and / or adding one to several amino acids in an amino acid sequence obtainable from a publicly known database. "An amino acid sequence obtained by deleting, inserting, substituting, and / or adding one to several amino acids" means that a sufficient number of amino acids (preferably 10 or fewer, more preferably 7 or fewer, and even more preferably 6 or fewer) have been deleted, inserted, substituted, and / or added by a publicly known method for producing mutant peptides, such as site-directed mutagenesis. Examples of substantially identical amino acid sequences include amino acid sequences that have an identity of at least 80% or more, preferably at least 85% or more, more preferably at least 90% or more, even more preferably at least 92% or more, particularly preferably at least 95% or more, and most preferably at least 99% or more, with an amino acid sequence obtainable from a publicly known database.

[0060] <<Dissolved Oxygen Partial Pressure Step (A1)>> In step (A1), the dissolved oxygen partial pressure of the culture medium in contact with the organoids is 20% or less, and may be 0.1% or more and 5.0% or less, 0.5% or more and 4.5% or less, or 1.0% or more and 4.0% or less.

[0061] The oxygen partial pressure in the medium in contact with the organoid can be calculated based on the value measured by a dissolved oxygen concentration meter, such as OXYGEN NANOPROBES (PYROSCIENC).

[0062] <Culture Medium> The culture medium for culturing organoids may be, for example, a liquid culture medium or a culture medium containing an extracellular matrix (ECM).

[0063] In the liquid culture medium step (A1), any medium may be used to culture the organoids as long as the effects of the present invention are achieved, and may be, for example, a liquid culture medium. A secretory factor secreted from the second organ may be added to the liquid culture medium.

[0064] In step (A1), when organoids are cultured in a liquid medium, the dissolved oxygen partial pressure of the liquid medium that contacts with the organoids is 20% or less, and may be 0.1% or more and 5.0% or less, or 0.5% or more and 4.5% or less, or 1.0% or more and 4.0% or less. The oxygen partial pressure (%) in the atmosphere that contains the liquid medium is 20% or less, and may be 0.1% or more and 6.5% or less, or 1.0% or more and 5.0% or less, relative to 100 kPa (100%).

[0065] In this specification, the atmosphere containing the culture medium or the like means, for example, the atmosphere inside an incubator containing the culture medium or the like during culture.

[0066] As a liquid medium for culturing organoids, it is preferable to use a medium for culturing vascular cells (e.g., vascular endothelial cells), a medium for culturing undifferentiated cells that differentiate into the first organ, or a mixture of these two media.

[0067] The culture medium for vascular cells may be, for example, a known culture medium for vascular endothelial cells. As the culture medium for vascular endothelial cells, it is preferable to use one containing at least one of hEGF (recombinant human epidermal growth factor), VEGF (vascular endothelial growth factor), hydrocortisone, bFGF, ascorbic acid, IGF1, FBS, antibiotics (e.g., gentamicin, amphotericin B, etc.), heparin, L-glutamine, phenolred, and BBE.

[0068] Examples of media that can be used for culturing vascular cells include KBM VEC-1 (manufactured by Kohjin Bio Co., Ltd.), EGM-2 Bullet Kit (manufactured by Lonza), EGM Bullet Kit (manufactured by Lonza), VascuLife EnGS CompKit (manufactured by LCT), Human Endothelial-SFM Basal Growth Medium (manufactured by Invitrogen), and Human Microvascular Endothelial Cell Growth Medium (manufactured by TOYOBO).

[0069] A known culture medium can be used as a culture medium for undifferentiated cells that differentiate into a first organ. When the undifferentiated cells are undifferentiated hepatocytes, the culture medium preferably contains at least one of ascorbic acid, BSA-FAF, insulin, hydrocortisone, and GA-1000.

[0070] Media for culturing undifferentiated hepatocytes include DMEM (Wako) containing dexamethasone (0.1 μM; Sigma-Aldrich, St. Louis, MO), oncostatin M (20 ng / mL; R&D Systems, Minneapolis, MN), and fetal bovine serum (5%), hepatocyte culture medium (HCM) (Cambrex, Baltimore, MD), HCM Bullet Kit (Lonza) without hEGF (recombinant human epidermal growth factor), and RPMI 1640 (Sigma-Aldrich) containing 1% B27 Supplements (GIBCO) and 10 ng / mL For example, one containing hHGF (Sigma-Aldrich) can be used.

[0071] As a liquid medium for culturing liver organoids, for example, a medium in which growth factors, differentiation factors, etc. described in conventional methods (Takebe et al., Massive and Reproducible Production of Liver Buds Entirely from Human Pluripotent Stem Cells, Cell Rep. 2017 21, 2661-2670; Supplemental Information) have been added to the basal medium described below can be used. The liquid medium for culturing liver organoids preferably contains one or more compounds selected from dexamethasone and oncostatin M. In the liquid medium, the concentration of dexamethasone is preferably 1 nM to 1 μM, more preferably 10 nM to 1000 nM, and even more preferably 20 nM to 500 nM. In the liquid medium, the concentration of Oncostatin M is preferably 0.1 to 1000 ng / mL, more preferably 1 to 200 ng / mL, and even more preferably 5 to 100 ng / mL.

[0072] The liquid medium for culturing renal organoids may be, for example, a medium prepared by adding growth factors, differentiation factors, etc., described in conventional methods (Shi M et al., Human ureteric bud organoids recapitulate branching morphogenesis and differentiate into functional collecting duct cell types, Nat Biotechnol. 2023 Feb;41(2):252-261.) to the basal medium described below. The liquid medium for culturing renal organoids is preferably a medium prepared by adding GDNF, FGF10, a GSK3 inhibitor, a BMP inhibitor, a TGF-β inhibitor, retinoic acid, and a MAP kinase inhibitor to the basal medium.

[0073] The liquid medium for culturing intestinal organoids may be, for example, a medium prepared by adding growth and differentiation factors, etc., described in a conventional method (Spence JR et al., "Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro," Nature. 2011 Feb. 3; 470(7332): 105-109.) to the basal medium described below. The liquid medium for culturing intestinal organoids is preferably a basal medium supplemented with R-spondin, Noggin, and EGF.

[0074] As a liquid medium for culturing brain organoids, for example, a medium in which growth and differentiation factors, etc. described in conventional methods (Mayhew CN and Singhania R., A review of protocols for brain organoids and applications for disease modeling, STAR Protoc. 2023 Mar 17; 4(1): 101860.) have been added to the basal medium described below can be used.

[0075] Alternatively, in step (A1), a medium containing extracellular matrix (hereinafter also referred to as ECM) may be used as the medium for culturing the organoids. The medium containing ECM is usually prepared by adding ECM or the like to a basal medium.

[0076] Culture medium containing ECM—extracellular matrix (ECM) The extracellular matrix is ​​a substance that serves as a scaffold for cells in cell culture. Examples of components of the extracellular matrix include components contained in basement membranes and glycoproteins present in intercellular spaces. Examples of components contained in basement membranes include type IV collagen, laminin, heparan sulfate proteoglycan, and entactin. Examples of glycoproteins present in intercellular spaces include collagen, laminin, entactin, fibronectin, fibrinogen, and heparin sulfate. The ECM may be natural, synthetic, or a mixture of natural and synthetic ECMs. Examples of commercially available ECM-containing culture media include Matrigel (a Corning product) and human laminin (Sigma product).

[0077] ... Basal medium In this specification, examples of basal medium include Dulbecco's modified Eagle's medium (DMEM), basal medium (MEM), knockout-DMEM (KO-DMEM), Glasgow essential medium (G-MEM), Basal Eagle's medium (BME), αMEM medium, DMEM / Ham's F12, Advanced DMEM / Ham's F12, Iscove's modified Dulbecco's medium, Ham's F-10, Ham's F-12, 199 medium, RPMI1640 medium, and mixed media thereof.

[0078] The medium containing the ECM may be supplemented with secretory factors secreted from the second organ.

[0079] In step (A1), the organoid may be cultured in contact with ECM. When the organoid is cultured in contact with ECM, it is preferable to culture the organoid embedded in a medium containing ECM.

[0080] The thickness of the culture medium containing ECM may be, for example, 0.50 mm to 30 mm, 1.00 mm to 20 mm, or 1.00 mm to 10 mm.

[0081] In step (A1), when the organoids are embedded in a medium containing ECM and cultured, the dissolved oxygen partial pressure of the medium containing ECM in contact with the organoids is 20% or less, 0.1% or more and 5.0% or less, 0.5% or more and 4.5% or less, or 1.0% or more and 4.0% or less, relative to 100 kPa (100%). The oxygen partial pressure (%) in the atmosphere containing the medium containing ECM may be 0.1% or more and 40.0% or less, 1.0% or more and 35.0% or less, 5.0% or more and 30.0% or less, or 10.0% or more and 25.0% or less, relative to 100 kPa (100%).

[0082] Alternatively, in step (A1), the organoids may be embedded in a medium containing ECM, and an overlay medium may be overlaid on the medium containing ECM to culture the organoids.

[0083] Layer medium: Layer medium is usually prepared by adding various components to a basal medium. For example, the liquid medium described above may be used as the layer medium. Examples of the basal medium include those described above for the medium containing ECM. Secretory factors secreted from the second organ may be added to the layer medium.

[0084] When culturing liver organoids, the overlay medium preferably contains one or more selected from Dexamethasone and Oncostatin M. In the overlay medium, the concentration of Dexamethasone is preferably 1 nM to 1 μM, more preferably 10 nM to 1000 nM, and even more preferably 20 nM to 500 nM. In the overlay medium, the concentration of Oncostatin M is preferably 0.1 to 1000 ng / mL, more preferably 1 to 200 ng / mL, and even more preferably 5 to 100 ng / mL.

[0085] When culturing intestinal organoids, examples of the layer medium include the liquid media described above as the liquid medium for culturing intestinal organoids.

[0086] When culturing renal organoids, examples of the overlay medium include the liquid media described above for culturing renal organoids.

[0087] The thickness of the ECM-containing medium may be, for example, 0.50 mm to 30 mm, 1.00 mm to 20 mm, or 1.00 mm to 10 mm. The thickness of the overlay medium may be, for example, 0.10 mm to 30 mm, 0.50 mm to 20 mm, or 1.00 mm to 10 mm. The thickness of the overlay medium relative to the thickness of the ECM-containing medium may be, for example, 0.1 to 10, or 0.3 to 3, as a ratio expressed as thickness of overlay medium / thickness of medium containing ECM.

[0088] In step (A1), when the organoids are embedded in a medium containing ECM and cultured by layering a medium containing ECM, the dissolved oxygen partial pressure of the medium containing ECM in contact with the organoids is 20% or less, 0.1% or more and 5.0% or less, 0.5% or more and 4.5% or less, or 1.0% or more and 4.0% or less, relative to 100 kPa (100%). The oxygen partial pressure (%) in the atmosphere containing the medium containing ECM and the layered medium may be 0.1% or more and 40.0% or less, 1.0% or more and 35.0% or less, 5.0% or more and 30.0% or less, or 10.0% or more and 25.0% or less, relative to 100 kPa (100%).

[0089] When organoids are embedded in a medium containing ECM and then cultured by layering a medium containing ECM on top of the medium, the dissolved oxygen partial pressure of the medium containing ECM that comes into contact with the organoids can be calculated as follows.

[0090] According to Fig. 2 in Kakni et al. Hypoxia-tolerant apical-out intestinal organoids to model host-microbiome interactions, J Tissue Eng. 2023 Jan-Dec; 14: 20417314221149208. doi: 10.1177 / 20417314221149208, when the oxygen partial pressure of the atmosphere containing the suspension culture medium (liquid medium) is 21%, the dissolved oxygen partial pressure of the liquid medium is approximately 17%. When the oxygen partial pressure of the atmosphere containing the liquid medium is 5%, the dissolved oxygen partial pressure of the liquid medium is approximately 4%. From these data, it can be seen that the dissolved oxygen partial pressure (Y １ %) is the oxygen partial pressure (X ) of the atmosphere containing the liquid medium. １ %) can be calculated as follows: １ = 0.8125X １ −0.0625 (1)

[0091] According to Kakni et al.'s Differentiation of pluripotent stem cells toward small intestinal organoids (page 3, left column), Fig. 2, when DMEM medium is layered on top of Matrigel in an atmosphere with an oxygen partial pressure of 21%, the dissolved oxygen partial pressure of Matrigel is 4%. In other words, when the oxygen partial pressure of the atmosphere containing the medium is 21%, the dissolved oxygen partial pressure of the liquid medium is 17%, and the dissolved oxygen partial pressure of Matrigel is 4%. From these data, it can be seen that when the medium is contained in the same atmosphere, the dissolved oxygen partial pressure of Matrigel (Y ２ %) is the dissolved oxygen partial pressure (Y １ %) can be calculated as follows: ２ = 4Y １ / 17 (2)

[0092] From the above formulas (1) and (2), the dissolved oxygen partial pressure of Matrigel (Y ２ %) is the oxygen partial pressure (X) of the atmosphere containing the medium. １ %) can be calculated as follows: ２ = 0.1912X１ −0.0147 (3)

[0093] According to the above formula (3), when the oxygen partial pressure of the atmosphere containing the culture medium is 40%, the dissolved oxygen partial pressure of Matrigel is 7.6%. When the oxygen partial pressure of the atmosphere containing the culture medium is 80%, the dissolved oxygen partial pressure of Matrigel is 15.3%. Even when a culture medium containing ECM other than Matrigel is used, the dissolved oxygen partial pressure of the ECM-containing culture medium can be estimated using the above formula (3).

[0094] In step (A1), the culture temperature is preferably 30 to 40°C, more preferably about 37°C.

[0095] In step (A1), the culture period is preferably 1 to 21 days, more preferably 2 to 14 days, and even more preferably 3 to 7 days.

[0096] <Step (A2)> In step (A2), the organoids cultured in step (A1) are cultured.

[0097] <<Dissolved Oxygen Partial Pressure Step (A2)>> In step (A2), the dissolved oxygen partial pressure of the culture medium in contact with the organoids is 20% or less relative to 100 kPa (100%), and may be 5.0% or more and 20.0% or less, 7.0% or more and 16.0% or less, 6.0% or more and 12.0% or less, or 6.0% or more and 10.0% or less.

[0098] In step (A2), the secretory factor secreted from the second organ may or may not be contacted with the organoid.The culture medium for culturing the organoid in step (A2) can be exemplified as the culture medium for culturing the organoid in step (A1).The culture medium in step (A2) can or does not need to be added with the secretory factor secreted from the second organ.

[0099] In step (A2), when organoids are cultured in a liquid medium, the dissolved oxygen partial pressure of the liquid medium that comes into contact with the organoids can be 5.0% or more and 20.0% or less, 7.0% or more and 16.0% or less, 6.0% or more and 12.0% or less, or 6.0% or more and 10.0% or less. The oxygen partial pressure (%) in the atmosphere that contains the liquid medium can be 6.0% or more and 25.0% or less, or 9.0% or more and 20.0% or less, relative to 100 kPa (100%).

[0100] In step (A2), when the organoids are embedded in a medium containing ECM and cultured, the dissolved oxygen partial pressure of the medium containing ECM in contact with the organoids may be 5.0% to 20.0%, 7.0% to 16.0%, 6.0% to 12.0%, or 6.0% to 10.0%. The oxygen partial pressure (%) in the atmosphere containing the medium containing ECM may be 10% to 100% relative to 100 kPa (100%), 25% to 90%, 30% to 70%, or 30% to 60%.

[0101] In step (A2), when the organoids are embedded in a medium containing ECM and cultured by layering a medium containing ECM, the dissolved oxygen partial pressure of the medium containing ECM in contact with the organoids may be 5.0% to 20.0%, 7.0% to 16.0%, 6.0% to 12.0%, or 6.0% to 10.0%. The oxygen partial pressure (%) in the atmosphere containing the medium containing ECM and the layered medium may be 10% to 100% relative to 100 kPa (100%), 25% to 90%, 30% to 70%, or 30% to 60%.

[0102] In step (A2), the culture temperature is preferably 30 to 40°C, more preferably about 37°C.

[0103] In step (A2), the culture period is preferably 1 to 30 days, more preferably 7 to 21 days.

[0104] The method for producing organoids according to the first aspect may include, prior to step (A1), a step of producing organoids from undifferentiated cells, mesenchymal cells, and vascular cells that differentiate into the first organ. Examples of the step of producing organoids include the methods described above.

[0105] In steps (A1) and (A2), the carbon dioxide partial pressure (%) in the atmosphere containing the culture medium for culturing organoids can be appropriately set by a person skilled in the art, and may be, for example, 1 to 10% or 2 to 7% relative to 100 kPa (100%).

[0106] In steps (A1) and (A2), the atmosphere containing the culture medium for culturing the organoids may have the partial pressures of oxygen and carbon dioxide as described above, or the remainder other than oxygen and carbon dioxide may be nitrogen.

[0107] As described above, in the method for producing organoids according to the first aspect, organoids from a first organ are contacted with secretory factors secreted from a second organ, and then cultured under hypoxic conditions, and then the organoids from the first organ are cultured under hyperoxic conditions. These steps can increase the proportion of undifferentiated cells in the organoids and increase the size of the organoids. Furthermore, it is possible to further enhance the function of the organoids.

[0108] (Method for producing organoids: Second aspect) The method for producing organoids according to the second aspect includes a step (B1) of contacting IL1α with organoids produced from undifferentiated cells that differentiate into liver, mesenchymal cells, and vascular cells, and culturing them.

[0109] In the second aspect, examples of the biological species of organs into which organoids can be differentiated include the same biological species as those in the first aspect.

[0110] In the second embodiment, the organoid to be cultured is prepared from undifferentiated cells that differentiate into liver, mesenchymal cells, and vascular cells. The undifferentiated cells that differentiate into liver, mesenchymal cells, and vascular cells are the same as those described above in the first embodiment.

[0111] The form and concentration of IL1α in step (B1) are the same as those of IL1α in step (A1) of the first embodiment.

[0112] The medium for culturing the organoids in step (B1) can be the medium suitable for culturing liver organoids described above in step (A1) of the first embodiment.

[0113] In step (B1), the dissolved oxygen partial pressure of the culture medium in contact with the organoid and the oxygen partial pressure of the atmosphere containing the culture medium may be those described above in step (A1).

[0114] The culture temperature and culture period in step (B1) may be the same as those in step (A1) of the first embodiment.

[0115] The method for producing organoids according to the second aspect preferably includes a step (B2) of culturing the organoids cultured in the step (B1).

[0116] The medium for culturing the organoids in step (B2) can be the medium suitable for culturing liver organoids described above in step (A1) of the first embodiment.

[0117] In step (B1), the dissolved oxygen partial pressure of the medium in contact with the organoids is preferably lower than the dissolved oxygen partial pressure of the medium in contact with the organoids in step (B2).

[0118] In step (B2), the dissolved oxygen partial pressure of the culture medium in contact with the organoid and the oxygen partial pressure of the atmosphere containing the culture medium may be those described above in step (A2) of the first aspect.

[0119] The culture temperature and culture period in step (B2) may be the same as those in step (A2) of the first embodiment.

[0120] The method for producing organoid according to the second aspect can also comprise the step (B1) before the step of producing liver organoid from undifferentiated cells, mesenchymal cells and vascular cells that differentiate into liver.The step of producing liver organoid can be, for example, the above-mentioned method.

[0121] As described above, according to the method for producing organoids according to the second aspect, by contacting liver organoids with IL1α and culturing them, the proportion of undifferentiated cells in the liver organoids can be increased, and the size of the liver organoids can be increased. Furthermore, the function of the liver organoids can be further improved.

[0122] (Organoid: Third Aspect) The organoid according to the third aspect is an organoid produced by the method for producing an organoid according to the first or second aspect. The organoid according to the third aspect is larger in size and more functionally superior than conventional organoids.

[0123] (Organoid: fourth aspect) In the organoid according to the fourth aspect, the proportion of the number of HNF4α positive and Ki67 positive cells in the organoid is 1% or more, preferably 1.5% or more, more preferably 2% or more, and even more preferably 3% or more relative to the number of HNF4α positive cells (100%).The upper limit of this proportion is not particularly limited, but may be 30%, may be 20%, or may be 10%.By making this proportion equal to or greater than the lower limit, it becomes easier to supply constantly differentiated hepatocytes, and when a part of the organoid is damaged, it becomes easier to supply differentiated hepatocytes.

[0124] The organoid according to the fourth aspect may be produced by the method for producing an organoid according to the second aspect.

[0125] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0126] (Materials and Methods) <Materials> Ff01 human iPS cells were used (Center for iPS Cell Research and Application, Kyoto University (CiRA)). Ff01 cells were passaged on dishes coated with Laminin 511 E8 fragment (iMatrix-511, Nippi) using StemFit (registered trademark) AK02N (Ajinomoto). The use of human iPS cells was approved by the University of Tokyo and Yokohama City University (approval number: A150924001).

[0127] <Method> Preparation of HE / EC / MC Hepatic endoderm cells (HE), vascular cells (endothelial cells, EC), and mesenchymal stem cells (MC) were prepared from human iPS cells using the methods described by Takebe et al. (Cell Rep 21, 2661-2670 (2017)) and Krumm J et al. (Cell Rep 38, 110604 (2022)). Hepatic endoderm cells are undifferentiated cells that differentiate into the liver.

[0128] KO-HE, prepared by the following procedure, was used as HE. The retroviral vector pGCDNsam-HNF4a-IRES-KO1, in which an IRES and kusabira orange (KO1) were ligated downstream of the HNF4a genomic fragment, was transfected into 293gp and 293gpg packaging cells, and viral particle production was induced using a tetracycline induction system. This viral vector was introduced into human iPS cells by infection with the culture supernatant of retrovirally infected cells to obtain KO-iPS cells. Next, the KO-iPS cells were induced to differentiate to generate cells expressing HNF4α and KO1 (hereinafter sometimes referred to as KO-HE). KO-HE are undifferentiated cells that differentiate into the liver and are sometimes referred to as "liver progenitor cells."

[0129] Organoid production: Liver organoids were produced using HE, EC, and MC differentiated from human iPS cells. Organoids were produced according to the following procedure, based on a conventional method (Takebe et al., Massive and Reproducible Production of Liver Buds Entirely from Human Pluripotent Stem Cells, Cell Rep. 2017 21, 2661-2670).

[0130] A cell population was prepared with a cell number ratio of 10:7:1 between HE, EC, and MC. These cells were resuspended in DMEM (Wako) / KBM VEC1 (Kohjin-Bio) (volume ratio 1:1) containing dexamethasone (0.1 μM; Sigma-Aldrich, St. Louis, MO), oncostatin M (20 ng / mL; R&D Systems, Minneapolis, MN), and fetal bovine serum (2.5%). To match the size of mouse fetal liver, the cell suspension was placed in 96-well PrimeSurface® plates (Sumitomo) and cultured for 3 days to obtain organoids.

[0131] An ECM-containing medium (volume ratio 1:1) consisting of a collagen solution [DMEM containing 1 μg / mL Rat collagen I (Cultrex) and 1% 1N NaOH (1%)] and Matrigel (Thermofisher) was prepared for culturing organoids. Next, 50 μL of ECM-containing medium was added to a 96-well PrimeSurface® plate and allowed to gel. Next, 50 μL of ECM-containing medium and the organoids were added to the well, and the organoids were embedded in the gel. Next, a layer culture medium (volume ratio 1:1) consisting of DMEM (2.5% FBS, 100 nM Dexamethasone, 20 ng / mL Oncostatin M) and VEC1 (plus supplement without FBS) was prepared. Next, 100 μL of layer culture medium was layered into the wells. Through the above procedure, a 96-well plate was layered with ECM-containing medium (1.5 mm thick), ECM-containing medium containing organoids (1.5 mm thick), and layer culture medium (3 mm thick) in order from the bottom of the well to prepare a culture medium for culturing organoids. If necessary, IL1α was added to the layer culture medium. The added IL1α had the amino acid sequence set forth in SEQ ID NO: 1. The added IL1α had the same sequence as mature IL1α, and was obtained by protease cleavage of the N-terminus of precursor IL1α. The added IL1α can be expressed by the base sequence shown in SEQ ID NO:2.

[0132] Colony assay: Cells were isolated from organoids using collagenase, pronase, and DNase. The obtained cells were cultured in Matrigel for 3 days, and the number of colonies formed from hepatic endoderm cells (KO-HE) expressing Kusabira-Orange was counted according to colony size. Small colonies were 100 μm ２ The larger colonies are less than 100 μm ２ That was all.

[0133] Whole-mount immunostaining and clearing. Organoids cultured overnight at 4°C were fixed with 4% PFA and then washed with 0.1% Tween-PBS. CHAPS was then used to enhance antibody permeability to the sample tissue. The samples were then incubated at 37°C for 1-2 hours, washed with 0.1% Tween-PBS, blocked with Protein Block (Agilent), and incubated at 4°C for 4 days in Protein Block-PBS (1:10 dilution) containing the primary antibody. The samples were then washed and reacted with the secondary antibody. The anti-Ki67 antibody used was ab15580 (Abcam), and the anti-HNF4α antibodies used were PP-H1415-00 and PP-K9218-00 (R&D Systems). A6684 (Sigma-Aldrich) was used as the anti-human albumin antibody.

[0134] Quantification of albumin secretion: 24 hours after medium exchange, the culture supernatant was collected and analyzed using a Human Albumin ELISA Kit (ab179887) at 1 × 10 ６ The amount of albumin secreted (ng) per day by each hepatic progenitor cell (HE) was calculated.

[0135] Analysis of Expression of Liver Progenitor Cell Markers and Hepatocyte Markers After culture, the organoids were dispersed to isolate liver progenitor cells, and the expression levels of each marker were analyzed by RNAseq analysis.

[0136] (Experimental Example 1) The period when blood flows from the placenta to the liver and the period when the liver of a mouse fetus is oxygenated were analyzed.

[0137] After injecting an anti-CD31 antibody that recognizes vascular cells into the umbilical vein, we analyzed the liver tissue of mouse fetuses. ＋ No vascular cells were observed, and CD31 ＋ Vascular cells were observed. This result indicates that blood flows from the placenta into the liver of mouse fetuses from E10.5 onwards.

[0138] Pimonidazole, a hypoxia marker, was used to analyze the timing of liver oxygenation in mouse fetuses. The results are shown in Figures 1A and 1B. Figure 1A shows the results of pimonidazole staining in the liver at E9.5 to E11.5. Figure 1B shows the percentage of pimonidazole-positive areas in the liver at E9.5 to E11.5 (one-way ANOVA, Tukey's test, E9.5 vs. E10.5, P = 0.9388; E9.5 vs. E11.5, P < 0.0001). At E10.5, blood flow into the liver was confirmed, but the liver was in a hypoxic state. Furthermore, the liver became oxygenated from E11.5 onward.

[0139] We searched for growth factors whose expression increases in the placenta after E10.5, when blood begins to flow into the liver of mouse fetuses, and discovered IL1α.

[0140] Experimental Example 2: Organoids were produced using human iPS cell-derived hepatic endoderm cells (HE), mesenchymal stem cells (MC), and endothelial cells (EC). IL1α was added to these organoids under hypoxic conditions, and the effect on organoid size was examined.

[0141] Organoids were formed using HE, MC, and EC according to the method described above in "Materials and Methods." The organoids were then embedded in a medium containing ECM, and IL1α was added to the overlay medium. As a control, IL1α was not added to the overlay medium. The final concentration of IL1α was 10 ng / mL. After the addition of IL1α, the organoids were cultured for 7 days. The area of ​​HE expressing Kusabira-Orange (KO-HE) and the volume of the organoids were then measured. KO-HE are undifferentiated cells that differentiate into the liver and are sometimes referred to as "liver progenitor cells."

[0142] The atmosphere in the incubator containing the ECM-containing medium and overlay medium was set to a normal atmospheric environment (i.e., 100 kPa total pressure, 20% oxygen tension). The oxygen tension in the ECM-containing medium in contact with the organoids was estimated to be approximately 4.0% relative to 100 kPa (100%). The oxygen tension in the ECM-containing medium was estimated based on Fig. 2 in Kakni et al. Hypoxia-tolerant apical-out intestinal organoids to model host-microbiome interactions, J Tissue Eng. 2023 Jan-Dec; 14: 20417314221149208. doi: 10.1177 / 20417314221149208.

[0143] The results are shown in Figures 2A to 2C. "Day 0" and "Day 7" in Figure 2A show the results of observing undifferentiated liver cells (KO-HE) under a fluorescence microscope. In Figure 2A, "Day 0" is a photograph taken immediately after the addition of IL1α, and "Day 7" is a photograph taken 7 days after the addition of IL1α. Ki67 shows the results of immunostaining for the proliferative cell marker Ki67, and HNF4α shows the results of immunostaining for the liver progenitor cell marker HNF4α. Figure 2B shows the maximum cross-sectional area of ​​KO-HE (n = 4; IL1α versus control Mann-Whitney test p-value = 0.0286). Figure 2C shows the results of measuring the volume of organoids (n = 4; IL1α versus control Mann-Whitney test p-value = 0.0286).

[0144] As shown in Figures 2A to 2C, it was confirmed that adding IL1α under hypoxic conditions facilitates the proliferation of liver progenitor cells and increases the size of organoids.

[0145] (Experimental Example 3) A colony assay was performed on the organoids obtained in Experimental Example 2 and cultured with the addition of IL1α. The results are shown in Figures 3A to 3C. Figure 3A is a photograph of an example of the obtained colony. In Figure 3A, "Day 0" is a colony obtained from organoids immediately after the start of culture, and "Day 3" is a colony obtained from organoids three days after the start of culture. Figure 3B is a graph showing the number of small colonies and large colonies. Figure 3C is a graph showing the number of small colonies and large colonies obtained from organoids cultured with IL1α, a marker for undifferentiated liver cells (ALB). ＋ CK19 ＋ ) is a photograph of cells expressing "ALB" and "CK19" is a photograph of cells expressing "ALB" and "CK19".

[0146] As shown in Figure 3A, larger colonies were observed in the IL1α-treated group than in the IL1α-free group. Large colonies are derived from more proliferative and less differentiated progenitor cells than small colonies.

[0147] As shown in Figure 3B, there was no significant difference in the number of small colonies between the IL1α-treated and IL1α-untreated groups (One-way ANOVA, Tukey's test, p = 0.3600), but there was a significant difference in the number of large colonies (One-way ANOVA, Tukey's test, p = 0.0019). It was confirmed that cells differentiating into more undifferentiated livers proliferated in the IL1α-treated organoids compared to the IL1α-untreated organoids.

[0148] As shown in Figure 3C, in the group to which IL1α was added, a marker of liver progenitor cells (ALB) ＋ CK19 ＋ )-expressing cells were identified.

[0149] (Experimental Example 4) Organoids were cultured in PrimeSurface plates (Sumitomo) using 200 μL of DMEM (Wako) / KBM VEC1 (Kohjin-Bio) (volume ratio 1:1) containing fetal bovine serum (2.5%). Organoids were prepared by adding IL1α to the organoids at three final concentrations: 1 ng / mL, 3.3 ng / mL, and 10 ng / mL. The results are shown in Figures 4A and 4B. Figure 4A is a photograph of the resulting organoids. Figure 4B is a graph showing the results of measuring the area of ​​the resulting organoids.

[0150] The results shown in Figures 4A and 4B indicate that the organoid size tended to increase with increasing concentration in the range of 1 to 3.3 ng / mL. No difference in organoid size was observed in the range of 3.3 to 10 ng / mL.

[0151] (Experimental Example 5) Organoids were cultured under the same conditions as in Experimental Example 2, with the addition of IL1α (early culture). The oxygen partial pressure in the incubator was then adjusted, and the organoids were cultured for an additional 7 days (late culture). The composition of the medium for the late culture was the same as that for the early culture, except that IL1α was not added. In the late culture, the volume of the overlay medium was 150 μL.

[0152] In the later stage of culture, the oxygen tension in the incubator containing the ECM-containing medium and the overlay medium was set to 20%, 40%, or 80%. In all cases, the total pressure in the incubator was 100 kPa.

[0153] As mentioned above, according to Fig. 2 of Kakni et al., the dissolved oxygen tension (Y ２ %) is the oxygen partial pressure (X ) of the atmosphere containing the ECM-containing medium and the overlay medium. １ %) can be calculated as follows: ２ = 0.1912X １-0.0147 (3) According to the above formula (3), when the oxygen partial pressure in the incubator was 20%, 40%, and 80%, the dissolved oxygen partial pressure in the medium containing ECM with which the organoids were in contact was estimated to be 4.0%, 7.6%, and 15.3%, respectively.

[0154] The results are shown in Figures 5A to 5B. Figure 5A shows a photograph of the obtained organoid. In Figure 5A, "Day 7" is a photograph of liver progenitor cells of organoids after early culture, and "Day 14" is a photograph of liver progenitor cells of organoids after late culture. Figure 5B is a graph showing the ratio of the area of ​​liver progenitor cell region of organoids after late culture to the liver progenitor cell region of organoids after early culture. Here, liver progenitor cells refer to cells that express KO.

[0155] In the later stage of culture, it was confirmed that the area of ​​the liver progenitor cell region was larger when the oxygen partial pressure in the incubator was 40% than when the oxygen partial pressure in the incubator was 20% or 80%. When comparing the oxygen partial pressure of 20% with the oxygen partial pressure of 40%, one-way ANOVA, Tukey's test, p=0.0339, and when comparing the oxygen partial pressure of 20% with the oxygen partial pressure of 80%, p=0.0121.

[0156] (Experimental Example 6) Organoids were produced under the following four conditions. Hereinafter, "oxygen partial pressure in the incubator" refers to the oxygen partial pressure (%) in the incubator containing the ECM-containing medium and the overlay medium relative to 100 kPa (100%). "Early culture" refers to days 0 to 7 of culture, and "late culture" refers to days 7 to 14 of culture. In the above culture and late culture, the medium composition was the same as in Experimental Example 2, except that IL1α was added or not added. When IL1α was added, the concentration of IL1α in the overlay medium was 10 ng / mL. In the late culture, the volume of the overlay medium was 150 μL.

[0157] In Test Example 1, the oxygen partial pressure in the incubator was set to 20% during early culture and late culture. IL1α was not added during either early culture or late culture. In Test Example 2, the oxygen partial pressure in the incubator was set to 20% during early culture and late culture. IL1α was added during early culture, but not during late culture. In Test Example 3, the oxygen partial pressure in the incubator was set to 20% during early culture, but not during late culture. IL1α was not added during early culture or late culture. In Test Example 4, the oxygen partial pressure in the incubator was set to 20% during early culture, but not during late culture. IL1α was added during early culture, but not during late culture. The experimental procedures for each test example are shown in Figure 6A.

[0158] Using the above formula (3), when the oxygen partial pressure in the incubator was 20% and 40%, the dissolved oxygen partial pressure in the culture medium containing the ECM in contact with the organoids was estimated to be 4.0% and 7.6%, respectively.

[0159] The results are shown in Figures 6B to 6G. Figure 6B is a photograph of the obtained organoids. "Day 0" is a photograph of the organoids before early culture in Test Examples 1 to 4. "Day 14" is a photograph of the organoids after late culture in Test Examples 1 to 4. The photographs of Ki67 and HNF4α are the results of immunostaining analysis of expression in organoids after late culture in Test Examples 1 to 4. Ki67 is a marker for proliferating cells. HNF4α (hepatocyte nuclear factor 4 alpha) is a marker for liver progenitor cells.

[0160] FIG. 6C shows the HNF4α expression in the organoids after the late culture in Test Examples 1 to 4. ＋ Ki67 relative to the number of cells ＋ HNF4α ＋1 is a graph showing the proportion of cells in the liver progenitor cell population. In comparing the proportion of cells, the n number for each test example was 3 or 4, the p value between test example 1 and test example 4 was 0.0008, the p value between test example 2 and test example 4 was 0.0072, and the p value between test example 3 and test example 4 was 0.0005. Statistical analysis was performed using one-way ANOVA and Tukey's test. In test example 4, it was confirmed that the proportion of cells with proliferative properties was higher in the population of liver progenitor cells than in test examples 1 to 3.

[0161] Figure 6D shows the results of analyzing the expression levels of liver progenitor cell markers AFP (α-fetoprotein), CPM (Carboxypeptidase M), DLK1 (Delta-like protein 1), and CDH2 (cadherin 2). In Test Example 4, compared to Test Example 1, it was confirmed that the expression levels of AFP, CPM, and DLK1 were high and the expression level of CDH2 was low. That is, in Test Example 4, compared to Test Example 1, it was suggested that the organoids contained more cells with an expression profile similar to that of liver progenitor cells.

[0162] 6E is a graph showing the results of measuring the volume of organoids after late culture. In comparing the volume of organoids, the n number of each test example was 4 or 6, the p value between test example 1 and test example 4 was 0.0002, the p value between test example 2 and test example 4 was 0.0010, and the p value between test example 3 and test example 4 was 0.0437, and statistical analysis was performed using one-way ANOVA and Tukey's test. In test example 4, compared to test examples 1 to 3, it was confirmed that the size of the organoids after late culture was larger.

[0163] 6F is a graph showing the results of measuring the amount of human albumin secreted in organoids after the late culture in Test Examples 1 and 4. It was confirmed that the amount of albumin secreted was higher in Test Example 4 than in Test Example 1 (n=3, Student's t-test, p=0.0479).

[0164] 6G shows the results of analyzing the expression levels of hepatocyte markers FABP1, H19, AHSG, ALB, SERPINA1, FGL1, TF, AGT, FGA, FGB, FGG, FN1, GPC3, TTR, APOC1, RBP4, APOA2, and APOC3. That is, in Test Example 4, it was suggested that the organoids contained more cells with an expression profile similar to that of hepatocytes than in Test Example 1.

[0165] It was found that culturing organoids under hypoxic conditions with the addition of IL1α in the early stage and under hyperoxic conditions in the later stage could increase their size and improve their function.

[0166] The present invention can be suitably used for creating human organs using iPS cells.

Claims

A step (A1) of contacting an organoid prepared from undifferentiated cells, mesenchymal cells, and vascular cells that differentiate into a first organ with a secretory factor secreted from a second organ and culturing the organoid; and (A2) culturing the organoid cultured in the step (A1), The dissolved oxygen partial pressure of the medium in contact with the organoid in step (A1) and the dissolved oxygen partial pressure of the medium in contact with the organoid in step (A2) are 20% or less relative to 100 kPa (100%); A method for producing organoids, wherein the dissolved oxygen partial pressure of the culture medium in contact with the organoids in step (A1) is lower than the dissolved oxygen partial pressure of the culture medium in contact with the organoids in step (A2). 　 The method for producing organoids described in claim 1, wherein the first organ is a liver. 　 The method for producing organoids described in claim 1, wherein the second organ is a placenta. 　 The method for producing organoids described in claim 2, wherein the secreted factor is IL1α. 　 A method for producing organoids, comprising the step of contacting IL1α with organoids produced from undifferentiated cells that differentiate into liver, mesenchymal cells, and vascular cells, and culturing the organoids. 　 An organoid produced by the organoid production method described in any one of claims 1 to 5. 　 An organoid in which the ratio of the number of cells that are HNF4α-positive and Ki67-positive to the number of cells that are HNF4α-positive (100%) is 1% or more.

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