Method for producing an organoid

Abstract

A production method for an organoid, the method comprising a step for culturing adult stem cells or a cellular tissue fragment containing adult stem cells in a medium containing a chimeric fibroblast growth factor (FGF) including a partial region of FGF1 and a partial region of FGF2. An organoid manufactured by the production method. A medium containing the chimeric FGF, wherein the contained amount of the chimeric FGF is 50 ng / mL or less. An evaluation method for a substance to be tested. By using the chimeric FGF, it is possible to reduce the contained amounts of growth factors contained in a medium.

Description

Technical Field

[0001] The present invention relates to a method for producing an organoid. More specifically, the present invention relates to a method for producing an organoid, a culture medium, an organoid, a method for evaluating a test substance, and a chimerized FGF. This application claims priority based on Japanese Patent Application No. 2019-226710 filed in Japan on December 16, 2019, the content of which is incorporated herein by reference.

Background Art

[0002] An organoid is a cultured cell formed by cell aggregation and has a structure and function similar to those of an organ in vivo. In recent years, research has been actively conducted to produce various organoids from somatic stem cells, stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells). For example, brain organoids, intestinal organoids, liver organoids, kidney organoids, stomach organoids, lung organoids, ovarian cancer organoids, biliary tract cancer organoids, etc. have been produced.

[0003] The production of organoids from stem cells is carried out by controlling signal transduction pathways to induce the proliferation, differentiation, etc. of stem cells. The MAPK signal transduction pathway is known to be a signal transduction pathway that controls the proliferation, growth, differentiation, transformation, and apoptosis, etc. of stem cells. Also, the MAPK signal transduction pathway is known to be downregulated by growth factors such as basic fibroblast growth factor (Fibroblast growth factor 2, FGF2) and epidermal growth factor (EGF). These growth factors are usually added components to the culture medium for producing organoids from stem cells (see Patent Document 1 and Non-Patent Document 1).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Non-Patent Literature

[0005]

Non-Patent Literature 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] By the way, in the production of organoids from stem cells, due to the inactivation of medium components, it is necessary to periodically replace the medium during cell culture. In particular, when organoids are used for regenerative medicine, drug screening, etc., organoids of sufficient size are required, and accordingly, the number of medium replacements increases. On the other hand, growth factors such as FGF2 and EGF are very costly and also have a risk of containing endotoxins and the like. For this reason, it is desired to reduce the amount of growth factors added to the medium. Therefore, an object of the present invention is to provide a technique for producing organoids that can reduce the content of growth factors contained in the medium.

Means for Solving the Problems

[0007] The present invention includes the following embodiments. [1] A method for producing organoids, comprising the step of culturing adult stem cells or a cell tissue piece containing adult stem cells in a medium containing a chimeric FGF containing a partial region of Fibroblast growth factor (FGF) 1 and a partial region of FGF2. [2] The production method according to [1], wherein the content of the chimeric FGF contained in the medium is 50 ng / mL or less. [3] The production method according to [1] or [2], wherein the medium further contains Epidermal Growth Factor (EGF). [4] The manufacturing method according to [3], wherein the total content of the chimeric FGF and the EGF contained in the medium is 100 ng / mL or less. [5] The chimeric FGF is a protein consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4, or consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4, and by containing 50 ng / mL or less in the medium Adult stem cells or Adult It is a protein having an activity capable of producing an organoid from a cell tissue piece containing stem cells, and is the manufacturing method according to any one of [1] to [4]. [6] The manufacturing method according to any one of [1] to [5], wherein the medium further contains an Insulin-like growth factor (IGF) signal transduction pathway enhancer. [7] The manufacturing method according to any one of [1] to [6], wherein the medium further contains a Transforming Growth Factor-β (TGF-β) signal transduction pathway inhibitor. [8] The manufacturing method according to any one of [1] to [7], wherein the medium further contains a Wnt signal transduction pathway enhancer. [9] The manufacturing method according to any one of [1] to [8], wherein the medium further contains a Rho kinase (ROCK) signal transduction pathway inhibitor.

[10] The manufacturing method according to any one of [1] to [9], wherein the medium further contains a Bone morphogenetic protein (BMP) signal transduction pathway inhibitor.

[11] A medium containing chimeric FGF, wherein the content of the chimeric FGF is 50 ng / mL or less.

[12] The medium according to claim 11, further containing EGF, wherein the total content of the chimeric FGF and the EGF is 100 ng / mL or less.

[13] An organoid produced by the method according to any one of [1] to

[10] . A method for evaluating a test substance, comprising: a step of bringing the organoid described in

[14]

[13] into contact with the test substance; and a step of evaluating the influence of the test substance on the organoid.

[15] It is a protein consisting of the amino acid sequence described in any one of SEQ ID NO: 1 to SEQ ID NO: 4, or consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence described in any one of SEQ ID NO: 1 to SEQ ID NO: 4, and by containing 50 ng / mL or less in the medium Adult stem cells or Adult a chimeric FGF, which is a protein having the activity of producing an organoid from a cell tissue piece containing stem cells.

Advantages of the Invention

[0008] According to the present embodiment, it is possible to provide a technique for producing an organoid capable of reducing the content of growth factors contained in the medium.

Brief Description of the Drawings

[0009]

Figure 1

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Figure 7

Embodiments for Carrying Out the Invention

[0010] Hereinafter, the present invention will be described in more detail by showing embodiments, but the present invention is not limited to the following embodiments at all.

[0011] Each component exemplified in this specification, for example, the components contained in the medium and the components used in each step, can be used alone or in combination of two or more, unless otherwise specified.

[0012] In this specification, the notation representing a numerical range such as "A to B" is synonymous with "A or more and B or less", and A and B are included in the numerical range.

[0013] In this specification, "a medium containing substance X" and "in the presence of substance X" mean a medium to which an exogenous substance X is added, a medium containing an exogenous substance X, or in the presence of an exogenous substance X. That is, when the cells or tissues present in the medium endogenously express, secrete, or produce the substance X, the endogenous substance X is distinguished from the exogenous substance X, and a medium that does not contain the exogenous substance X, even if it contains the endogenous substance X, does not fall within the category of "a medium containing substance X".

[0014] [Method for producing organoids] One embodiment includes a step of culturing adult stem cells or a cell tissue piece containing adult stem cells in a medium containing a chimeric FGF (hereinafter also referred to as "FGFC") containing a partial region of Fibroblast growth factor (FGF) 1 and a partial region of FGF2. This is a method for producing organoids.

[0015] By the production method of the present embodiment, in the production of organoids, the content of the growth factor added to the medium can be reduced. The production method of the present embodiment is particularly suitable for the production of organoids such as intestinal organoids, liver organoids, ovarian cancer organoids, lung organoids, and gastric organoids.

[0016] In the production of organoids, the reason why the content of growth factors contained in the medium could be reduced by containing chimeric FGF in the medium is presumed as follows. That is, there is a specific fibroblast growth factor receptor (FGFR) that reacts with FGF, and it is known that there are 7 types including isoforms. By chimerizing FGF, it becomes possible to react with many types of FGFRs, and as a result, it is presumed that it can be cultured even at a low concentration. In particular, epithelial stem cells, which are important in the culture of adult stem cells, are said to highly express FGFR2b, but FGF2 does not have good reactivity with FGFR2b. On the other hand, it is known that FGF1 can react with all 7 types of FGFRs including FGFR2b, and FGFC, which has both the properties of FGF2 and FGF1, can react with FGFR2b, and as a result, it is presumed that it can be cultured even at a low concentration.

[0017] Adult stem cells, also called somatic stem cells, tissue stem cells, or cancer stem cells, are undifferentiated cells that exist in the body of a living organism. Usually, adult stem cells have the ability to differentiate into specific multiple types of cells. In the production method of this embodiment, the adult stem cells may be cultured and maintained cells or primary cells existing in tissues excised from an adult. Further, epithelial stem cells are preferable as adult stem cells.

[0018] In this specification, a cell mass in which cells are aggregated is referred to as a cell aggregate. Among cell aggregates, in particular, those containing adult stem cells and cells differentiated from adult stem cells and having a structure and function similar to those of organs in the living body are referred to as organoids.

[0019] In the production method of the present embodiment, as the culture medium, a medium obtained by adding chimeric FGF to a basal medium can be used. Examples of the basal medium include BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM (GMEM) medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, F-12 medium, DMEM / F12 medium, IMDM / F12 medium, Ham's medium, RPMI 1640 medium, and Fischer's medium. As the basal medium, a medium obtained by mixing these media may also be used.

[0020] The chimeric FGF in the production method of the present embodiment is a fusion protein containing a partial region of FGF1 and a partial region of FGF2. Examples of the NCBI accession numbers of human FGF1 protein include NP_001244138.1, NP_001244137.1, NP_001341881.1, etc. Examples of the NCBI accession numbers of mouse FGF1 protein include NP_034327.1, etc. Examples of the NCBI accession numbers of human FGF2 protein include NP_001348594.1, NP_001997.5, etc. Examples of the NCBI accession numbers of mouse FGF2 protein include NP_032032.1, etc.

[0021] As the chimeric FGF, a protein consisting of the amino acid sequence described in any one of SEQ ID NOs: 1 to 4 is preferable. By containing the chimeric FGF in the medium at 50 ng / mL or less Adult stem cells or Adult As long as it has the activity to produce organoids from cell tissue pieces containing stem cells, it may have mutations with respect to the protein consisting of the amino acid sequence described in any one of SEQ ID NOs: 1 to 4.

[0022] That is, the chimeric FGF may be a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, or added in the amino acid sequence described in any one of SEQ ID NOs: 1 to 4. Here, one or several means 1 to 10, preferably 1 to 5, more preferably 1 to 3, still more preferably 1 to 2.

[0023] In the amino acid sequence of the chimeric FGF, the amino acid corresponding to the 43rd amino acid in the amino acid sequence of SEQ ID NO: 1 is preferably glutamine, valine, isoleucine, or leucine.

[0024] The chimeric FGF in which the amino acid corresponding to the 43rd amino acid in the amino acid sequence of SEQ ID NO: 1 is glutamine, valine, isoleucine, or leucine has high heat resistance and is stable as compared with FGF1 and FGF2.

[0025] Among them, the chimeric FGF in which the amino acid corresponding to the 43rd amino acid in the amino acid sequence of SEQ ID NO: 1 is isoleucine has a particularly high melting temperature (Tm) and high heat resistance.

[0026] The amino acid corresponding to the 43rd amino acid in the amino acid sequence of SEQ ID NO: 1 can be identified, for example, by aligning the target amino acid sequence with the amino acid sequence of SEQ ID NO: 1 using an alignment program such as ClustalW.

[0027] Conventionally, in the production of organoids, it was usual to add about 100 ng / mL of FGF2 to the culture medium. In contrast, by using chimeric FGF instead of FGF2, the content of the chimeric FGF contained in the culture medium can be reduced. The content of the chimeric FGF contained in the culture medium is usually 50 ng / mL or less, preferably 20 ng / mL or less, more preferably 15 ng / mL or less, still more preferably 10 ng / mL or less, and particularly preferably 5 ng / mL or less.

[0028] In the production method of the present embodiment, the culture medium may further contain Epidermal Growth Factor (EGF). When the culture medium contains EGF, the total content of chimeric FGF and EGF contained in the culture medium can be reduced. When the culture medium contains EGF, the total content of chimeric FGF and EGF contained in the culture medium is usually 100 ng / mL or less, preferably 70 ng / mL or less.

[0029] The NCBI accession number of human EGF protein is NP_001171601.1 etc.

[0030] In the production method of the present embodiment, the culture medium preferably further contains a Wnt signaling pathway enhancer.

[0031] Examples of the Wnt signaling pathway enhancer include Wnt family members, R-spondin, Norrin, GSK-3β inhibitors, etc. Among these, Wnt family members and R-spondin are preferred.

[0032] Examples of Wnt family members include Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, and Wnt16 etc.

[0033] As the Wnt family member, Wnt3a or Wnt4 is preferred, and Wnt3a is more preferred. Wnt3a is preferably a complex with Afamin in order to improve stability.

[0034] When using Wnt3a as the Wnt signaling pathway enhancer, the concentration of Wnt3a contained in the culture medium is usually 10 ng / mL to 10 μg / mL, preferably 10 ng / mL to 1 μg / mL, for example 100 ng / mL to 700 ng / mL.

[0035] Examples of R-spondin include R-spondin 1, R-spondin 2, R-spondin 3, R-spondin 4, etc.

[0036] Examples of the NCBI accession numbers of human R-spondin 1 protein include NP_001229838.1, XP_006710646.1, etc. Examples of the NCBI accession numbers of mouse R-spondin 1 protein include NP_619624.2, etc. Examples of the NCBI accession numbers of human R-spondin 2 protein include XP_011515320.1, XP_011515321.1, NP_848660.3, etc. Examples of the NCBI accession numbers of mouse R-spondin 2 protein include NP_766403.1, etc. Examples of the NCBI accession numbers of human R-spondin 3 protein include XP_016866867.1, XP_016866868.1, NP_116173.2, etc. Examples of the NCBI accession numbers of mouse R-spondin 3 protein include NP_082627.3, etc. Examples of the NCBI accession numbers of human R-spondin 4 protein include NP_001035096.1, NP_001025042.2, etc. Examples of the NCBI accession numbers of mouse R-spondin 4 protein include NP_001035779.1, etc.

[0037] When using R-spondin as a Wnt signaling pathway enhancer, the concentration of R-spondin contained in the medium is usually 10 ng / mL to 10 μg / mL, preferably 10 ng / mL to 1 μg / mL, more preferably 100 ng / mL to 500 ng / mL.

[0038] Examples of GSK-3β inhibitors include CHIR99021 (CAS number: 252917-06-9), Kenpaullone (CAS number: 142273-20-9), and 6-Bromoindirubin-3'-oxime (BIO, CAS number: 667463-62-9), etc.

[0039] When using a GSK-3β inhibitor as an enhancer of the Wnt signaling pathway, the concentration of the GSK-3β inhibitor contained in the medium is usually 0.1 μM to 10 μM.

[0040] In the production method of the present embodiment, it is preferable that the medium further contains an enhancer of the Insulin-like growth factor (IGF) signaling pathway.

[0041] Examples of the IGF signaling pathway enhancer include IGF-1, IGF-2, etc., and IGF-1 is preferable. Examples of the NCBI accession numbers of human IGF-1 protein include NP_001104753.1, NP_001104755.1, etc. Examples of the NCBI accession numbers of mouse IGF-1 protein include NP_001104745.1, etc. Examples of the NCBI accession numbers of human IGF-2 protein include NP_001007140.2, NP_001278790.1, etc. Examples of the NCBI accession numbers of mouse IGF-2 protein include NP_034644.2, NP_001302418.1, etc.

[0042] In the production method of the present embodiment, it is preferable that the medium further contains an inhibitor of the Transforming Growth Factor-β (TGF-β) signaling pathway.

[0043] TGF-β signaling pathway inhibitors are substances that inhibit the signal transduction pathway transmitted by the Smad family. Examples of TGF-β signaling pathway inhibitors include A83-01 (CAS No.: 909910-43-6), SB-431542 (CAS No.: 301836-41-9), SB-505124 (CAS No.: 694433-59-5), SB-525334 (CAS No.: 356559-20-1), LY364947 (CAS No.: 396129-53-6), SC-203294 (CAS No.: 627536-09-08), SD-208 (CAS No.: 627536-09-8), SJN2511 (CAS No.: 446859-33-2), etc. Among these, A83-01 and SB-431542 are preferred.

[0044] The concentration of the TGF-β signaling pathway inhibitor contained in the medium is usually 10 nM to 100 μM, preferably 100 nM to 10 μM.

[0045] In the production method of this embodiment, the medium preferably further contains a Rho kinase (ROCK) signaling pathway inhibitor.

[0046] Examples of ROCK signaling pathway inhibitors include, for example, Y-27632 (CAS No.: 129830-38-2), Fasudil / HA1077 (CAS No.: 105628-07-7), H-1152 (CAS No.: 871543-07-6), Wf-536 (CAS No.: 539857-64-2), and derivatives thereof.

[0047] The concentration of the ROCK signaling pathway inhibitor contained in the medium is usually 0.1 μM to 100 μM, preferably 0.1 μM to 50 μM, more preferably 0.1 μM to 30 μM.

[0048] In the production method of this embodiment, the medium preferably further contains a Bone morphogenetic protein (BMP) signaling pathway inhibitor.

[0049] Examples of BMP signal transduction pathway inhibitors include Noggin, cordin, follistatin, dorsomorphin (CAS No.: 866405-64-3), DMH1 (CAS No.: 1206711-16-1), LDN193189 (CAS No.: 1062368-24-4), etc.

[0050] The concentration of the BMP signal transduction pathway inhibitor contained in the medium is usually 0.5 μM to 10 μM. When Noggin is used as the BMP signal transduction pathway inhibitor, the concentration of Noggin contained in the medium is usually 10 ng / mL to 1000 ng / mL, preferably 10 ng / mL to 500 ng / mL, more preferably 10 ng / mL to 300 ng / mL.

[0051] In the production method of this embodiment, the medium may further contain, as other additives, a medium supplement, an antibacterial agent, serum, a serum substitute, insulin, albumin, etc.

[0052] Examples of the medium supplement include a supplement for culturing nerve cells such as the product "B-27 Serum-Free Supplement" (Thermo Fisher Scientific), a glutamine-containing supplement such as the product "GlutaMax" (Thermo Fisher Scientific) containing L-glutamine, L-alanyl L-glutamine, etc., an aqueous amino acid solution such as "MEM Non-Essential Amino Acids Solution" (Thermo Fisher Scientific), and 2-mercaptoethanol.

[0053] Examples of the antibacterial agent include penicillin antibiotics, cephem antibiotics, macrolide antibiotics, tetracycline antibiotics, phosphomycin antibiotics, aminoglycoside antibiotics, and new quinolone antibiotics.

[0054] In the culture of adult stem cells or cell tissue pieces containing adult stem cells, the cells may be embedded in an extracellular matrix (ECM), or ECM may be added to the culture medium. Examples of ECM include components contained in the basement membrane and glycoproteins present in the intercellular space. Examples of components contained in the basement membrane include type IV collagen, laminin, heparan sulfate proteoglycan, entactin, etc. Examples of glycoproteins present in the intercellular space include collagen, laminin, entactin, fibronectin, heparin sulfate, etc. As the ECM, commercially available products containing ECM may be used. Examples of commercially available products containing ECM include Matrigel (registered trademark, Corning), human laminin (Sigma), etc.

[0055] Matrigel contains basement membrane components derived from Engelbreth Holm Swarm mouse sarcoma. The main components of Matrigel are type IV collagen, laminin, heparan sulfate proteoglycan, and entactin. In addition to these, various growth factors such as epidermal growth factor (EGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), insulin-like growth factor 1 (IGF-1), and TGF-β are included. There is also a grade of Matrigel with low concentrations of various growth factors, and the concentrations are less than 0.5 ng / mL for EGF, less than 0.2 ng / mL for NGF, less than 5 pg / mL for PDGF, less than 5 ng / mL for IGF-1, and less than 1.7 ng / mL for TGF-β. It is preferable to use a grade of Matrigel with a low content ratio of various growth factors.

[0056] In the culture of adult stem cells or cell tissue pieces containing adult stem cells, it is preferable to change the culture medium about once every 1 to 5 days. Also, when the formed organoids are large, it is preferable to disperse the organoids. Dispersion means separating the cells into a cell population of 10,000 or fewer cells by dispersion treatment such as enzymatic treatment or physical treatment. Dispersion can be performed by treating the organoids with a cell dispersion solution, etc.

[0057] Examples of the cell dispersion solution include solutions containing enzymes such as trypsin, collagenase, hyaluronidase, elastase, pronase, DNase, papain, etc., or chelating agents such as ethylenediaminetetraacetic acid. As the cell dispersion solution, commercially available cell dispersion solutions can also be used. Examples of commercially available cell dispersion solutions include TrypLE Select and TrypLE Express manufactured by Thermo Fisher Scientific.

[0058] [Culture medium] One embodiment is a culture medium containing chimeric FGF, wherein the content of the chimeric FGF is 50 ng / mL or less. By culturing adult stem cells or cell tissue pieces containing adult stem cells in the culture medium of this embodiment, organoids can be produced. Therefore, the culture medium of this embodiment can be said to be a culture medium for organoid production.

[0059] The culture medium of this embodiment is obtained by adding chimeric FGF to a basal medium. The basal medium and chimeric FGF are the same as those described above.

[0060] The culture medium of this embodiment preferably further contains EGF, and the total content of the chimeric FGF and the EGF is 100 ng / mL or less. EGF is the same as that described above.

[0061] The culture medium of this embodiment can further contain a Wnt signaling pathway enhancer, an IGF signaling pathway enhancer, a TGF-β signaling pathway inhibitor, a ROCK signaling pathway inhibitor, a BMP signaling pathway inhibitor, and other additives. Each of these components is the same as those described above.

[0062] [Organoid] 1 An embodiment is an organoid produced by the manufacturing method described above. Since the organoid of this embodiment can be manufactured at a reduced cost of growth factors and the like, it can be easily manufactured in large quantities at low cost. Therefore, for example, it can be suitably used for applications such as large-scale evaluation of test substances described later.

[0063] The organoid of this embodiment is manufactured using chimeric FGF instead of conventionally used FGF2. Therefore, there may be differences in gene expression profiles and the like between the organoid of this embodiment and the organoid manufactured by the conventional manufacturing method. However, since it requires a great deal of trial and error and labor to identify such differences, it is not realistic to identify the organoid of this embodiment by gene expression profiles and the like, and it is considered realistic to identify it by the manufacturing method.

[0064] [Method for Evaluating Test Substances] 1 An embodiment is a method for evaluating a test substance, including a step of bringing an organoid produced by the manufacturing method described above into contact with the test substance, and a step of evaluating the influence of the test substance on the organoid.

[0065] By the method of this embodiment, it is possible to evaluate a test substance using an organoid having a structure and function similar to those of cells in vivo. The method of this embodiment can be applied to screening of therapeutic drugs for various diseases and the like.

[0066] Examples of the test substance include a natural compound library, a synthetic compound library, an existing drug library, a metabolite library, and the like.

[0067] The influence of the test substance on the organoid can be evaluated, for example, at the gene level, protein level, and metabolite level. In addition, the influence of the test substance on the organoid can also be evaluated by evaluating the morphology of the organoid.

[0068] Evaluation at the gene level can be performed, for example, by RNA-seq analysis, DNA microarray analysis, real-time PCR, etc. Evaluation at the protein level can be performed, for example, by Western blotting, ELISA, immunostaining, etc. Evaluation at the metabolite level can be evaluated, for example, by liquid chromatography (LC), mass spectrometry (MS), LC / MS, etc. Evaluation of the morphology of organoids can be performed, for example, by microscopic observation, immunostaining, etc.

[0069] [Chimeric FGF] One embodiment is a protein consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4, or an amino acid sequence in which one or several amino acids are deleted, substituted, or added in the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4, and by containing 50 ng / mL or less in the medium Adult stem cells or Adult a chimeric FGF, which is a protein having an activity capable of producing organoids from cell tissue pieces containing stem cells.

[0070] As described later in the examples, by using the chimeric FGF of the present embodiment, the content of the growth factor added to the medium in the production of organoids can be reduced.

[0071] Also, as described above, in the amino acid sequence of chimeric FGF, the amino acid corresponding to the 43rd amino acid in the amino acid sequence of SEQ ID NO: 1 is preferably glutamine, valine, isoleucine, or leucine.

[0072] Among them, from the viewpoints of having high heat resistance and being stable, the amino acid corresponding to the 43rd amino acid in the amino acid sequence of SEQ ID NO: 1 is preferably valine, isoleucine, or leucine, and more preferably isoleucine.

Examples

[0073] Hereinafter, the present embodiment will be described more specifically based on examples, but the present embodiment is not limited to these examples.

[0074] [Experimental Example 1] (Production of Organoids) Adult stem cells were cultured using a medium containing FGF2 or chimeric FGF at various concentrations in the presence or absence of EGF, and the formation of organoids was examined.

[0075] 《Preparation of Medium》 As the basal medium, the product "Advanced DMEM / F12" (Thermo Fisher Scientific) was used, and a medium supplemented with the components described in Table 1 below was prepared. In Table 1, "w / o EGF formulation" means a medium formulation without EGF, and "w / EGF formulation" means a medium formulation with EGF.

[0076] As the medium supplement, B-27 serum-free supplement (Thermo Fisher Scientific) was used.

[0077] As FGF2, the product "FGF-Basic, Human, Recombinant, Animal Free" (PeproTech) was used. As the chimeric FGF, the product "FGFC" (FUJIFILM Wako Pure Chemical Corporation) was used.

[0078] As EGF, the product "Animal-Free Recombinant Murine EGF" (PeproTech) was used.

[0079] As the Wnt signal enhancer (Wnt signal transduction pathway enhancer), mouse R-spondin 1 conditioned medium (self-prepared) and the product "Afamin / Wnt3a CM" (MBL Life Science) were used.

[0080] As the insulin-like growth factor (IGF signal transduction pathway enhancer), the product name "Recombinant Human IGF-I / IGF-1 Protein, CF" (R&D Systems) was used.

[0081] As the TGF-β inhibitor (TGF-β signal transduction pathway inhibitor), A83-01 (TOCRIS Bioscience) was used.

[0082] As the ROCK inhibitor (ROCK signal transduction pathway inhibitor), the product name "Y-27632, MF" (FUJIFILM Wako Pure Chemical Corporation) was used.

[0083] As the BMP inhibitor (BMP signal transduction pathway inhibitor), mouse Noggin conditioned medium (self-prepared) was used.

[0084]

Table 1

[0085] 《Organoid Formation》 Colon tissue pieces were used as cell tissue pieces containing adult stem cells. More specifically, cells in which a reporter construct was introduced into exon 18 of the LGR5 locus of cells derived from colon tissue pieces by genome editing were used in the experiment. The LGR5 gene is a stem cell marker. In addition, IRES-tdTomato was used as the reporter construct. These cells express tdTomato, a fluorescent protein, when they express LGR5.

[0086] The above cells were embedded in 20 μL of Matrigel (registered trademark, Corning) per 1000 cells and seeded in a 48-well plate. Subsequently, Matrigel was allowed to stand at 37°C to solidify. Subsequently, the medium of each composition described above was added around Matrigel. Thereafter, the medium was changed every 2 - 3 days.

[0087] Subsequently, the expression of tdTomato was detected by fluorescence microscopy, and the expression of the stem cell marker LGR5 protein was evaluated.

[0088] Figures 1(a) to (d) are micrographs of organoids formed in the absence of EGF on the 9th day of culture. Figure 1(a) is a bright-field image of organoids formed in the presence of FGF2 at each concentration shown in the figure. Figure 1(b) is a fluorescence micrograph detecting the expression of LGR5 protein in the same field of view as Figure 1(a). Figure 1(c) is a bright-field image of organoids formed in the presence of FGFC at each concentration shown in the figure. Figure 1(d) is a fluorescence micrograph detecting the expression of LGR5 protein in the same field of view as Figure 1(c).

[0089] Figures 2(a) to (d) are micrographs of organoids formed in the presence of EGF on the 8th day of culture. Figure 2(a) is a bright-field image of organoids formed in the presence of FGF2 at each concentration shown in the figure. Figure 2(b) is a fluorescence micrograph detecting the expression of LGR5 protein in the same field of view as Figure 2(a). Figure 2(c) is a bright-field image of organoids formed in the presence of FGFC at each concentration shown in the figure. Figure 2(d) is a fluorescence micrograph detecting the expression of LGR5 protein in the same field of view as Figure 2(c).

[0090] As a result, in the absence of EGF, it was revealed that even when adult stem cells were cultured in a medium containing 10 ng / mL of FGF2, the formation of organoids was insufficient. On the other hand, it was revealed that organoids can be formed in a medium containing FGFC even in the absence of EGF. Also, as shown in Figures 1(c) and (d), it was revealed that organoids can be formed even when the concentration of FGFC in the medium is reduced to about 2.5 ng / mL.

[0091] Furthermore, it was revealed that in the presence of EGF, organoids can be formed by culturing adult stem cells in a medium containing 12.5 ng / mL of FGF2. Also, it was revealed that in the presence of EGF, organoids can be formed even when the concentration of FGFC in the medium is reduced to about 3.1 ng / mL.

[0092] Moreover, it was revealed that when using a medium containing FGFC, the number of colony formations is larger compared to the case of using a medium containing FGF2.

[0093] [Experimental Example 2] (Preparation of FGFC variants) A gene fragment encoding an FGFC variant was prepared by artificial synthesis. The amino acid sequence of FGFC is shown in SEQ ID NO: 1. As FGFC variants, a variant in which the 43rd glutamine residue of FGFC is substituted with valine (hereinafter referred to as "FGFC-V", and the amino acid sequence is shown in SEQ ID NO: 2), a variant in which the 43rd glutamine residue of FGFC is substituted with isoleucine (hereinafter referred to as "FGFC-I", and the amino acid sequence is shown in SEQ ID NO: 3), and a variant in which the 43rd glutamine residue of FGFC is substituted with leucine (hereinafter referred to as "FGFC-L", and the amino acid sequence is shown in SEQ ID NO: 4) were prepared.

[0094] Subsequently, the gene fragments encoding FGFC, FGFC-V, FGFC-I, and FGFC-L were respectively inserted into the expression vector pET-3a, expressed in BL21(DE3)pLysS, purified as proteins, and used in the following experiments.

[0095] [Experimental Example 3] (Production of organoids using FGFC variants) Adult stem cells were cultured using a medium containing FGFC or FGFC variants at various concentrations, and the formation of organoids was examined.

[0096] 《Preparation of medium》 As a basal medium, the medium prepared by adding the components described in Table 2 below using the product name "Advanced DMEM / F12" (Thermo Fisher Scientific) was used. In Table 2, "formulation w / o EGF" means a medium formulation without EGF, and "formulation w / EGF" means a medium formulation with EGF. As the FGFC and FGFC variants, those prepared in Experimental Example 2 were used. Also, as the EGF, Wnt signal enhancer, insulin-like growth factor, TGF-β inhibitor, ROCK inhibitor, and BMP inhibitor, the same ones as in Experimental Example 1 were used.

[0097]

Table 2

[0098] 《Production of Organoids》 As the cell tissue piece containing adult stem cells, the same cells as in Experimental Example 1 were used. First, the cells were embedded with 20 μL of Matrigel (registered trademark, Corning) per 1000 cells and seeded in a 48-well plate. Subsequently, the Matrigel was allowed to stand at 37°C to solidify. Subsequently, the media of each composition described above were added around the Matrigel. Thereafter, the medium was changed every 2 - 3 days.

[0099] Subsequently, the expression of tdTomato was detected by fluorescence microscopy observation, and the expression of the LGR5 protein, which is a stem cell marker, was evaluated.

[0100] Figures 3(a) - (d) are micrographs of each organoid on the 9th day of culture formed in the absence of EGF. Figure 3(a) is a bright-field image of the organoid formed in the presence of FGFC at each concentration shown in the figure. Figure 3(b) is a bright-field image of the organoid formed in the presence of FGFC-V at each concentration shown in the figure. Figure 3(c) is a bright-field image of the organoid formed in the presence of FGFC-I at each concentration shown in the figure. Figure 3(d) is a bright-field image of the organoid formed in the presence of FGFC-L at each concentration shown in the figure.

[0101] Figure 4(a) is a fluorescence microscopy photograph detecting the expression of LGR5 protein in the same visual field as in Figure 3(a). Figure 4(b) is a fluorescence microscopy photograph detecting the expression of LGR5 protein in the same visual field as in Figure 3(b). Figure 4(c) is a fluorescence microscopy photograph detecting the expression of LGR5 protein in the same visual field as in Figure 3(c). Figure 4(d) is a fluorescence microscopy photograph detecting the expression of LGR5 protein in the same visual field as in Figure 3(d).

[0102] Figures 5(a) to (d) are microscopy photographs of each organoid on the 8th day of culture formed in the presence of EGF. Figure 5(a) is a bright-field image of the organoid formed in the presence of FGFC at each concentration shown in the figure. Figure 5(b) is a bright-field image of the organoid formed in the presence of FGFC-V at each concentration shown in the figure. Figure 5(c) is a bright-field image of the organoid formed in the presence of FGFC-I at each concentration shown in the figure. Figure 5(d) is a bright-field image of the organoid formed in the presence of FGFC-L at each concentration shown in the figure.

[0103] Figure 6(a) is a fluorescence microscopy photograph detecting the expression of LGR5 protein in the same visual field as in Figure 5(a). Figure 6(b) is a fluorescence microscopy photograph detecting the expression of LGR5 protein in the same visual field as in Figure 5(b). Figure 6(c) is a fluorescence microscopy photograph detecting the expression of LGR5 protein in the same visual field as in Figure 5(c). Figure 6(d) is a fluorescence microscopy photograph detecting the expression of LGR5 protein in the same visual field as in Figure 5(d).

[0104] As a result, it was revealed that organoids can be produced to the same extent as when using FGFC, regardless of which FGFC variant is used. Also, as shown in Figures 3(a) to (d), Figures 4(a) to (d), Figures 5(a) to (d), and Figures 6(a) to (d), it was revealed that organoids can be produced even when the concentration of FGFC or FGFC variant in the medium is reduced to 2.5 ng / mL or 3.1 ng / mL.

[0105] [Experimental Example 4] (Examination of the heat resistance of FGFC variants) The heat resistance of FGFC, FGFC-V, FGFC-I, and FGFC-L prepared in Experimental Example 2 was examined. Also, for comparison, the heat resistance of FGF1 (product name "FGF-Acidic, Human, Recombinant", PeproTech) and FGF2 (product name "FGF-Basic, Human, Recombinant, Animal Free", PeproTech) was similarly examined.

[0106] For the examination of heat resistance, a commercially available kit (product name "Protein Thermal Shift TM Dye Kit", Thermo Fisher Scientific) was used. After preparing samples according to the kit manual, the heat resistance of each protein was examined using a real-time PCR device (product name "7500fast", Thermo Fisher Scientific). The measurement results were analyzed using software (product name "Protein Thermal Shift TM Software v1.3", Thermo Fisher Scientific).

[0107] Figure 7 is a graph showing the melting temperature (Tm) of each measured protein. The horizontal axis of the graph indicates temperature (°C). Also, the Tm values of each protein are shown in Table 3 below.

[0108]

Table 3

[0109] As a result, it was revealed that FGFC has higher heat resistance than FGF2. Also, it was revealed that FGFC-V, FGFC-I, and FGFC-L have even higher heat resistance than FGFC.

Industrial Applicability

[0110] According to this embodiment, it is possible to provide a technology for producing organoids that can reduce the content of growth factors contained in the medium.

Claims

1. A method for producing an organoid, comprising the step of culturing an adult stem cell or a tissue piece containing adult stem cells in a medium containing a chimeric FGF containing a partial region of Fibroblast growth factor (FGF) 1 and a partial region of FGF2, wherein the chimeric FGF is a protein consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 4, or consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 4, and the amino acid corresponding to the 43rd amino acid in the amino acid sequence of SEQ ID NO: 1 is valine, isoleucine or leucine.

2. The production method according to claim 1, wherein the content of the chimeric FGF contained in the medium is 50 ng / mL or less.

3. The production method according to claim 1 or 2, wherein the medium further contains Epidermal Growth Factor (EGF).

4. The production method according to claim 3, wherein the total content of the chimeric FGF and the EGF contained in the medium is 100 ng / mL or less.

5. The production method according to any one of claims 1 to 4, wherein the chimeric FGF is a protein having an activity capable of producing an organoid from an adult stem cell or a tissue piece containing adult stem cells by containing 50 ng / mL or less in the medium.

6. The production method according to any one of claims 1 to 5, wherein the medium further contains an Insulin-like growth factor (IGF) signaling pathway enhancer.

7. The production method according to any one of claims 1 to 6, wherein the medium further contains a Transforming Growth Factor-β (TGF-β) signaling pathway inhibitor.

8. The production method according to any one of claims 1 to 7, wherein the medium further contains a Wnt signaling pathway enhancer.

9. The production method according to any one of claims 1 to 8, wherein the medium further contains a Rho kinase (ROCK) signaling pathway inhibitor.

10. The production method according to any one of claims 1 to 9, wherein the medium further contains an inhibitor of the Bone morphogenetic protein (BMP) signaling pathway.

11. A medium containing chimeric FGF, wherein the chimeric FGF is a protein consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 4, or consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 4, and the amino acid corresponding to the 43rd amino acid in the amino acid sequence of SEQ ID NO: 1 is valine, isoleucine or leucine, and the content of the chimeric FGF is 50 ng / mL or less.

12. The medium according to claim 11, further containing EGF, wherein the total content of the chimeric FGF and the EGF is 100 ng / mL or less.

13. A step of producing an organoid by the production method according to any one of claims 1 to 10, a step of bringing the organoid produced by the above step into contact with a test substance, and a step of evaluating the influence of the test substance on the organoid, An evaluation method for a test substance, comprising:

14. A chimeric FGF which is a protein consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 4, or consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 4, and the amino acid corresponding to the 43rd amino acid in the amino acid sequence of SEQ ID NO: 1 is valine, isoleucine or leucine, and has an activity capable of producing an organoid from an adult stem cell or a cell tissue piece containing an adult stem cell by containing 50 ng / mL or less in a medium.

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