Culture medium for co-culturing microorganisms and animal cells and its use

Abstract

We provide technology for the stable co-culture of microorganisms and animal cells. [Solution] A culture medium for co-culturing microorganisms and animal cells containing 15 mM or more of a buffering agent having a pKa of 6.0 to 9.0 at 20°C, and from animal cells co-culturing with microorganisms, the ratio of 28S rRNA content to 18S rRNA content (28S rRNA / 18S rRNA) is 2 or more, or the absorbance at a wavelength of 260 nm (A 260 ) and absorbance at a wavelength of 280 nm (A 280 ) ratio (A 260 / A 280 A method for producing total RNA having a pKa of 2 or more, comprising the steps of co-culturing a microorganism and animal cells in a culture medium containing 15 mM or more of a buffer having a pKa of 6.0 to 9.0 at 20°C, and preparing total RNA from the animal cells.

Description

[Technical Field]

[0001] This invention relates to a culture medium for co-culturing microorganisms and animal cells, and to the use thereof. [Background technology]

[0002] Intestinal bacteria are not only involved in maintaining our health, but are also known to cause various diseases. Therefore, elucidating the interaction mechanisms between intestinal bacteria and host cells is attracting considerable attention worldwide.

[0003] To verify the detailed molecular functions of intestinal bacteria, it is necessary to establish an evaluation system for co-culturing intestinal bacteria and intestinal epithelial cells in vitro. The intestinal tract of animals is maintained in an anaerobic state, and most of the bacteria commensal there are anaerobic bacteria that can only grow under anaerobic conditions. In order to verify the physiological functions that these anaerobic bacteria exert on intestinal epithelial cells, it is necessary to culture intestinal epithelial cells under anaerobic conditions. However, epithelial cells, including intestinal epithelial cells, cannot be cultured under anaerobic conditions. Therefore, the inventors previously developed a technique to culture epithelial cells in a planar culture and easily control the oxygen partial pressure on the apical membrane side and the basement membrane side to culture them in an environment closer to that of a living organism (see, for example, Patent Document 1).

[0004] Incidentally, organoids are three-dimensional structures formed by the accumulation of cells, and they have structures and functions similar to organs in the body. In recent years, research into creating various organoids has been actively conducted. To date, for example, intestinal organoids, liver organoids, kidney organoids, stomach organoids, lung organoids, ovarian organoids, biliary tract organoids, pancreatic organoids, and brain organoids have been created. For example, Patent Documents 2 and 3 describe culture media for organoids. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] International Publication No. 2020 / 235206 [Patent Document 2] International Publication No. 2012 / 168930 [Patent Document 3] International Publication No. 2017 / 199811 [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] RNA-seq analysis is commonly used when studying the function of gut bacteria on intestinal epithelial cells. This requires the recovery of highly purified total RNA from intestinal epithelial cells co-cultured with gut bacteria.

[0007] In response, the inventors discovered that when intestinal epithelial cells were cultured in a conventional organoid culture medium in co-culture with intestinal bacteria, the intestinal epithelial cells were damaged, making it impossible to recover high-purity total RNA.

[0008] The present invention aims to provide a technology for stably co-culturing microorganisms and animal cells. According to the present invention, highly pure total RNA can be recovered from animal cells co-cultivated with microorganisms. [Means for solving the problem]

[0009] The present invention includes the following embodiments. [1] A culture medium for co-culturing microorganisms and animal cells, containing 15 mM or more of a buffer with a pKa of 6.0 to 9.0 at 20°C. [2] The buffering agent is 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris), N-(2-acetamide)iminodiacetic acid (ADA), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), N-(2-acetamide)-2-aminoethanesulfonic acid (ACES), 2-hydroxy-3-morpholinopropanesulfonic acid (MOPSO), N-[tris(hydroxymethyl)methyl]glycine (Tricine), N,N-bis(2-hydroxyethyl)glycine (Bicine), 2-[N,N-bis(2-hydroxyethyl)amino]-1- The culture medium according to [1], comprising tansulfonic acid (BES), 3-morpholinopropane-1-sulfonic acid (MOPS), N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), 3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid (TAPSO), piperazine-1,4-bis(2-hydroxypropanesulfonic acid) (POPSO), 4-(2-hydroxyethyl)piperazine-1-(2-hydroxypropane-3-sulfonic acid) (HEPPSO), 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS), tricine, bicine, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), glycinamide, or acetamidoglycine. [3] The culture medium according to [1] or [2], wherein the animal cells are epithelial cells. [4] From animal cells co-cultured with microorganisms, the ratio of 28S rRNA content to 18S rRNA content (28S rRNA / 18S rRNA) is 2 or greater, or the absorbance at a wavelength of 260 nm (A 260 ) and absorbance at a wavelength of 280 nm (A 280 ) ratio (A 260 / A 280) A method for producing all RNAs where the [[ID=]] is 2 or more, comprising the steps of co-culturing microorganisms and animal cells in a medium containing 15 mM or more of a buffer having a pKa of 6.0 to 9.0 at 20 °C, and preparing all RNAs from the animal cells.

Advantages of the Invention

[0010] According to the present invention, a technique for stably co-culturing microorganisms and animal cells can be provided. According to the present invention, highly pure all RNAs can be recovered from animal cells co-cultured with microorganisms.

Brief Description of the Drawings

[0011] [Figure 1] Figure 1 is a photograph and a schematic cross-sectional view explaining a culture vessel previously developed by the inventors. The upper left is a photograph taken of the culture vessel from the side. The upper center and the upper right are photographs taken of the state where the culture vessel is installed in a 24-well cell culture plate. The upper center is a photograph taken from above. The upper right is a photograph taken from the side. The lower part is a schematic cross-sectional view explaining the state of co-culturing epithelial cells and microorganisms using the culture vessel. [Figure 2] Figure 2 is an image showing the results of RNA analysis microchip electrophoresis in Experimental Example 1. [Figure 3] Figure 3 is a graph showing the results of quantifying the density of the bands in lanes 1 to 4 of Figure 2 in Experimental Example 1. [Figure 4] Figure 4 is a graph showing the results of quantifying the density of the bands in lanes 5 to 8 of Figure 2 in Experimental Example 1. [Figure 5] Figure 5 is a graph showing the results of calculating the ratio of the contents of 28S rRNA and 18S rRNA in all RNAs in Experimental Example 1. [Figure 6] Figure 6 is a graph showing the results of calculating the ratio (A260 / A280) of the absorbance at wavelength 260 nm (A260) and the absorbance at wavelength 280 nm (A280) of all RNAs in Experimental Example 1. [Figure 7]Figure 7 is a graph showing the results of measuring the total RNA mass extracted from monolayer cultured epithelial cells under each condition in Experimental Example 1. [Figure 8] Figure 8 shows photographs of Bifidobacterium longum colonies co-cultured with human cells in culture media containing HEPES at various concentrations in Experimental Example 2. [Figure 9] Figure 9 is a graph showing the colony-forming units of Bifidobacterium longum calculated in Experimental Example 2. [Figure 10] Figure 10 shows the results of RNA analysis microchip electrophoresis in Experimental Example 3. [Figure 11] Figure 11 is a graph showing the results of quantifying the intensity of the bands in lanes 1-4 of Figure 10 in Experimental Example 3. [Figure 12] Figure 12 is a graph showing the results of quantifying the intensity of the bands in lanes 5-8 of Figure 10 in Experimental Example 3. [Figure 13] Figure 13 is a graph showing the results of calculating the ratio of 28S rRNA and 18S rRNA content in total RNA in Experimental Example 3. [Figure 14] Figure 14 is a graph showing the results of calculating the ratio (A260 / A280) of the absorbance of total RNA at a wavelength of 260 nm (A260) and the absorbance at a wavelength of 280 nm (A280) in Experimental Example 3. [Figure 15] Figure 15 is a graph showing the results of measuring the total RNA concentration extracted from monolayer cultured epithelial cells under each condition in Experimental Example 3. [Figure 16] Figure 16 is a photograph showing colonies of Bifidobacterium longum co-cultured with human cells in culture media containing various concentrations of MES in Experimental Example 4. [Figure 17] Figure 17 is a graph showing the colony-forming units of Bifidobacterium longum calculated in Experimental Example 4. [Modes for carrying out the invention]

[0012] [Culture medium for co-culturing microorganisms and animal cells] In one embodiment, the present invention provides a culture medium for co-culturing microorganisms and animal cells, containing 15 mM or more of a buffering agent having a pKa of 6.0 to 9.0 at 20°C.

[0013] Typical organoid culture media contain 10 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES, CAS number: 7365-45-9) as a buffer.

[0014] However, as will be described later in the examples, the inventors found that when animal cells co-cultured with microorganisms in a conventional organoid culture medium, the animal cells are damaged, and it is not possible to recover highly pure total RNA.

[0015] In response to this, the inventors discovered that by co-culturing microorganisms and animal cells using a culture medium containing a buffer at a concentration of 15 mM or higher, it is possible to stably co-culture microorganisms and animal cells and recover highly pure total RNA from the animal cells, thus completing the present invention.

[0016] Examples of buffering agents with a pKa of 6.0 to 9.0 at 20°C include 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES, CAS number: 7365-45-9), 2-(N-morpholino)ethanesulfonic acid (MES, CAS number: 145224-94-8), and bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris, CA CAS No.: 6976-37-0), N-(2-acetamide)iminodiacetic acid (ADA, CAS No.: 26239-55-4), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES, CAS No.: 5625-37-6), N-(2-acetamide)-2-aminoethanesulfonic acid (ACES, CAS No.: 7365-82-4), 2-hydroxy-3-morpholinopropanesulfone Acid (MOPSO, CAS No.: 68399-77-9), N-[Tris(hydroxymethyl)methyl]glycine (Tricine, CAS No.: 5704-04-1), N,N-bis(2-hydroxyethyl)glycine (Bicine, CAS No.: 150-25-4), 2-[N,N-bis(2-hydroxyethyl)amino]-1-ethanesulfonic acid (BES, CAS No.: 10191-1) 8-1), 3-Morpholinopropane-1-sulfonic acid (MOPS, CAS number: 1132-61-2), N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES, CAS number: 7365-44-8), 3-[N-Tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid (TAPSO, CAS number: 68399-81-5), Piperazine-1,Examples include 4-bis(2-hydroxypropanesulfonic acid) (POPSO, CAS number: 68189-43-5), 4-(2-hydroxyethyl)piperazine-1-(2-hydroxypropane-3-sulfonic acid) (HEPPSO, CAS number: 68399-78-0), 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS, CAS number: 16052-06-5), tricine (CAS number: 5704-04-1), bicine (CAS number: 150-25-4), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS, CAS number: 29915-38-6), glycinamide (CAS number: 598-41-4), acetamidoglycine (CAS number: 7365-83-5), etc.

[0017] The concentration of the buffer in the culture medium in this embodiment is not particularly limited as long as it is a concentration that provides the effects of the present invention and does not adversely affect the growth of microorganisms and animal cells, and may be 15 mM or more, 20 mM or more, 30 mM or more, 40 mM or more, 50 mM or more, 60 mM or more, 70 mM or more, 80 mM or more, 90 mM or more, or 100 mM or more. Furthermore, the concentration of the buffer in the culture medium may be, for example, 200 mM or less, 190 mM or less, 180 mM or less, 170 mM or less, 160 mM or less, 150 mM or less, 140 mM or less, 130 mM or less, 120 mM or less, 110 mM or less, 100 mM or less, 90 mM or less, 80 mM or less, 70 mM or less, 60 mM or less, 50 mM or less, 40 mM or less, or 30 mM or less.

[0018] Examples of animal cells include cells derived from mammals. Mammals include, for example, rodents such as mice, rats, hamsters, and guinea pigs; lagomorphs such as rabbits; ungulates such as pigs, cattle, goats, horses, and sheep; carnivores such as dogs and cats; and primates such as humans, monkeys, rhesus macaques, crab-eating macaques, marmosets, orangutans, and chimpanzees.

[0019] Epithelial cells are preferred as animal cells. In this specification, epithelial cells include differentiated epithelial cells and epithelial stem cells obtained from epithelial tissue. "Epithelial stem cells" means cells that have long-term self-renewal function and the ability to differentiate into epithelial differentiated cells, and means stem cells derived from epithelial tissue. Examples of epithelial tissues include the cornea, oral mucosa, skin, conjunctiva, bladder, renal tubules, kidneys, digestive organs (esophagus, stomach, duodenum, small intestine (including jejunum and ileum), large intestine (including colon)), liver, bile ducts, pancreas, mammary glands, salivary glands, lacrimal glands, prostate gland, hair follicles, trachea, lungs, fallopian tubes, etc.

[0020] The epithelial cells may be epithelial cells obtained from epithelial tissue, cell lines established from epithelial cells, or epithelial cell-derived organoids. The epithelial cells may also be epithelial tumor cells that have become cancerous from the epithelial tissue-derived cells mentioned above.

[0021] Examples of cell lines established from epithelial cells include human colon cancer-derived cell lines such as Caco-2 cells, HT-29 cells, SW-480 cells, and LS-174T cells.

[0022] Examples of epithelial cell-derived organoids include intestinal organoids, liver organoids, kidney organoids, gastric organoids, lung organoids, ovarian organoids, biliary tract organoids, pancreatic organoids, salivary gland organoids, mammary gland organoids, endometrial organoids, bladder organoids, and prostate organoids. Epithelial cell-derived organoids may be derived from normal cells or from cancer cells.

[0023] Examples of microorganisms include those whose interactions with animal cells are analyzed. For instance, intestinal epithelial cells may be used as the animal cells, and intestinal bacteria as the microorganisms. Most intestinal bacteria are anaerobic bacteria that can only grow under anaerobic conditions.

[0024] The microorganisms used are not particularly limited; for example, bacteria belonging to the orders Clostridiales, Bacteroides, Bifidobacteriales, Vercomicrobiales, Desulfovibrioles, etc., can be used. One type of microorganism may be used alone, or two or more types may be used in combination.

[0025] Figure 1 shows a photograph and schematic cross-sectional view illustrating a culture vessel previously developed by the inventors. By using the culture vessel shown in Figure 1, epithelial cells can be cultured in a planar manner, and the oxygen partial pressure on the apical membrane side and the basement membrane side can be easily controlled, allowing for culture in an environment closer to that of a living organism. Furthermore, anaerobic bacteria and epithelial cells can be co-cultured.

[0026] The upper left image in Figure 1 is a photograph of the culture vessel taken from the side. The upper center and upper right images show the culture vessel placed on a 24-well cell culture plate. The upper center image is a photograph taken from above. The upper right image is a photograph taken from the side. The lower part of Figure 1 is a schematic cross-sectional view illustrating the co-culture of microorganisms and animal cells using the culture vessel. The animal cells may be epithelial cells.

[0027] The culture vessel comprises an upper container and a lower container, and the bottom surface of the upper container is fitted with a membrane that allows at least some components of the culture medium to pass through but not cells.

[0028] Epithelial cells are cultured inside the upper container, and once the epithelial cells reach confluence, the upper container becomes isolated, allowing the inside of the upper container to be maintained under anaerobic conditions. The cell membrane on the luminal side of the epithelial cell is called the apical membrane, and the cell membrane on the vascular side is called the basement membrane. The inside of the upper container faces the apical membrane side of the epithelial cell, and the inside of the lower container faces the basement membrane side of the epithelial cell.

[0029] When co-culturing microorganisms with epithelial cells, animal cell culture medium is placed in the lower container. As a result, oxygen is supplied to the basement membrane side of the epithelial cells through the membrane from the animal cell culture medium, allowing the epithelial cells to survive permanently. Then, by seeding anaerobic bacteria on the apical membrane side of the epithelial cells, contact can be brought with the epithelial cells without exposure to oxygen. As a result, epithelial cells and anaerobic bacteria can be co-culturified.

[0030] The upper container may contain animal cell culture media or bacterial culture media. If animal cell culture media is placed in the lower container, epithelial cells can be maintained even if the culture medium inside the upper container is replaced with bacterial culture media. Examples of bacterial culture media include modified GAM broth (Nissui Co., Ltd.), enhanced Clostridium medium, BHI medium, BL medium, LB medium, and EG medium.

[0031] The culture medium in the upper container (upper layer medium) contains 15 mM or more of a buffer with a pKa of 6.0 to 9.0 at 20°C. The culture medium in the lower container (lower layer medium) may or may not contain 15 mM or more of a buffer with a pKa of 6.0 to 9.0 at 20°C.

[0032] When the animal cells to be cultured are epithelial cell-derived organoids, an animal cell culture medium capable of maintaining stem cells contained in the epithelial cells in an undifferentiated state is preferably used. When the animal cells to be cultured are established cell lines, an animal cell culture medium suitable for that cell line should be used.

[0033] Culture media for epithelial cell-derived organoids include media containing at least one selected from the group consisting of insulin-like growth factor 1 (IGF1), fibroblast growth factor 2 (FGF2), and EGF-like growth factor, and at least one selected from the group consisting of Wnt agonists, bone morphogenetic factor (BMP) inhibitors, and transforming growth factor-β (TGF-β) inhibitors. The culture medium for epithelial cell-derived organoids is preferably serum-free.

[0034] Culture media for epithelial cell-derived organoids are media prepared by adding the above-mentioned components to a basic medium. Basic media include any serum-free cell culture medium. Examples of serum-free cell culture media include synthetic media buffered to pH 7.2-7.6. More specifically, examples include Advanced Dulbecco's Modified Eagle Medium / Ham F-12 (DMEM / F12) supplemented with glutamine, insulin, B27 supplement (Thermo Fisher Scientific), N-Acetyl-L-cysteine ​​(Wako Pure Chemical Industries), penicillin or streptomycin, and transferrin. Alternatively, RPMI 1640 medium (Roswell Park Memorial Institute 1640 medium), Advanced RPMI medium, etc., may be used as basic media.

[0035] The concentration of IGF1 in the culture medium for epithelial cell-derived organoids is not particularly limited, but is preferably 5 ng / mL to 1 μg / mL, more preferably 10 ng / mL to 1 μg / mL, and even more preferably 50 ng / mL to 500 ng / mL.

[0036] The concentration of FGF2 in the culture medium for epithelial cell-derived organoids is not particularly limited, but is preferably 5 ng / mL to 1 μg / mL, more preferably 10 ng / mL to 1 μg / mL, and even more preferably 50 ng / mL to 500 ng / mL.

[0037] Examples of EGF-like growth factors include EGF, EREG, and HBGF. The concentration of EGF in the culture medium for epithelial cell-derived organoids is not particularly limited, but is preferably 5 ng / mL to 1 μg / mL, more preferably 10 ng / mL to 1 μg / mL, and even more preferably 50 ng / mL to 500 ng / mL. EREG is an EGF-like growth factor that specifically binds to ErbB1 and ErbB4 among the tyrosine kinase (ErbB) family receptors (ErbB1 to 4). The concentration of EREG in the cell culture medium is not particularly limited, but is preferably 5 ng / mL to 1 μg / mL, more preferably 10 ng / mL to 1 μg / mL, and even more preferably 50 ng / mL to 500 ng / mL.

[0038] BMPs bind as dimeric ligands to receptor complexes consisting of two different receptor serine / threonine kinases, type I and type II receptors. The type II receptor phosphorylates the type I receptor, resulting in the activation of this receptor kinase. This type I receptor then phosphorylates a specific receptor substrate (SMAD), leading to transcriptional activity via a signaling pathway. Generally, BMP inhibitors are drugs that, for example, block or inhibit the binding of BMP molecules to BMP receptors, or bind to BMP molecules to form a complex that neutralizes BMP activity. Alternatively, BMP inhibitors can also act as antagonists or inverse agonists, for example, by binding to BMP receptors and blocking or inhibiting the binding of BMP molecules to the receptor.

[0039] BMP inhibitors preferably have inhibitory activity of 50% or more, more preferably 70% or more, even more preferably 80% or more, and particularly preferably 90% or more, compared to the BMP activity level in the absence of the inhibitor. BMP inhibitory activity can be evaluated, for example, by measuring the transcriptional activity of BMP.

[0040] The BMP inhibitors included in the culture medium for epithelial cell-derived organoids are preferably natural BMP-binding proteins, such as Noggin, Gremlin, Chordin, Chordin-like proteins such as Chordin Domain; Follistatin, Follistatin-related proteins such as Follistatin Domain; DAN-like proteins such as DAN, DAN-cysteine-knot Domain; Sclerostin / SOST, Decorin, α-2 macroglobulin, etc.

[0041] Among BMP inhibitors, Chordin-like proteins or DAN-like proteins are preferred, with Chordin-like proteins being more preferred. Among Chordin-like proteins, Noggin is preferred. Chordin-like proteins and DAN-like proteins are diffusible proteins that bind to BMP molecules with various degrees of affinity and can inhibit the approach of BMP molecules to signal transduction receptors.

[0042] The concentration of the BMP inhibitor in the culture medium for epithelial cell-derived organoids is preferably 10 to 100 ng / mL, more preferably 20 to 100 ng / mL, and even more preferably 50 to 100 ng / mL.

[0043] Transforming growth factor-β (TGF-β) is a type of growth factor produced in almost all cells, including those in the kidneys, bone marrow, and platelets. There are five subtypes of TGF-β (β1 to β5). TGF-β is known to promote the proliferation of osteoblasts and the synthesis and proliferation of connective tissue such as collagen, while suppressing the proliferation of epithelial cells and osteoclasts. Generally, TGF-β inhibitors are drugs that, for example, block or inhibit the binding of TGF-β to the TGF-β receptor, or bind to TGF-β to form a complex that neutralizes TGF-β activity. Alternatively, TGF-β inhibitors are drugs that, for example, bind to the TGF-β receptor and block or inhibit the binding of TGF-β to the receptor, acting as antagonists or inverse agonists.

[0044] The TGF-β inhibitor has inhibitory activity of preferably 50% or more, more preferably 70% or more, even more preferably 80% or more, and particularly preferably 90% or more, compared to the TGF-β activity level in the absence of the inhibitor.

[0045] Examples of TGF-β inhibitors included in the culture medium for epithelial cell-derived organoids include A83-01 (CAS number: 909910-43-6), SB-431542 (CAS number: 301836-41-9), SB-505124 (CAS number: 694433-59-5), SB-525334 (CAS number: 356559-20-1), LY364947 (CAS number: 396129-53-6), SD-208 (CAS number: 627536-09-8), and SJN2511 (CAS number: 446859-33-2), with A83-01 being the preferred choice.

[0046] The concentration of the TGF-β inhibitor contained in the culture medium for epithelial cell-derived organoids is preferably 100 nM to 10 μM, more preferably 500 nM to 5 μM, and even more preferably 500 nM to 2 μM.

[0047] In this specification, "Wnt agonist" means a drug that activates T-cell factor (hereinafter also referred to as TCF) / lymphoid enhancer factor (hereinafter also referred to as LEF)-mediated transcription within cells. Wnt agonists are not limited to Wnt family proteins, but include Wnt agonists that bind to and activate Frizzled receptor family members, inhibitors of intracellular β-catenin degradation, and TCF / LEF activators. Preferably, a Wnt agonist is at least one selected from the group consisting of Wnt proteins, R-spondin, and GSK-3β inhibitors.

[0048] As a Wnt agonist included in the culture medium for epithelial cell-derived organoids, a complex of Wnt protein and afamin is more preferred, and it is even more preferable that both the Wnt protein-afamin complex and R-spondin are included.

[0049] The Wnt protein is not particularly limited, and Wnt proteins from various organisms can be used. Among them, mammalian Wnt proteins are preferred. Examples of mammals include humans, mice, rats, cattle, pigs, and rabbits. Examples of mammalian Wnt proteins include Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, and Wnt16. Multiple types of Wnt proteins may be used in combination.

[0050] Methods for producing Wnt protein include, for example, using Wnt protein-expressing cells. The origin of the Wnt protein-expressing cells (species, culture form, etc.) is not particularly limited; any cell that stably expresses Wnt protein is acceptable, and even cells that transiently express Wnt protein are acceptable. Examples of Wnt protein-expressing cells include L cells that stably express mouse Wnt3a (ATCC CRL-2647) and L cells that stably express mouse Wnt5a (ATCC CRL-2814). Furthermore, Wnt protein-expressing cells can be produced using known genetic recombination techniques. Specifically, Wnt protein-expressing cells can be produced by inserting the DNA encoding the desired Wnt protein into a known expression vector and introducing the resulting expression vector into appropriate host cells. The base sequence of the gene encoding the desired Wnt protein can be obtained, for example, from a known database such as GenBank.

[0051] The Wnt protein expressed by Wnt protein-expressing cells may be a fragment of the Wnt protein, or it may contain amino acid sequences other than the Wnt protein amino acid sequence, as long as it possesses Wnt activity. There are no particular limitations on the amino acid sequences other than the Wnt protein amino acid sequence; for example, amino acid sequences of affinity tags can be used. Furthermore, the amino acid sequence of the Wnt protein does not need to be exactly the same as amino acid sequences obtained from known databases such as GenBank; as long as it possesses Wnt activity, it may be substantially the same as amino acid sequences obtained from known databases.

[0052] Examples of amino acid sequences that are substantially identical to the amino acid sequences of Wnt proteins obtained from publicly known databases such as GenBank include amino acid sequences in which one to several amino acids are deleted, substituted, or added compared to the amino acid sequences obtained from publicly known databases.

[0053] An amino acid sequence in which one to several amino acids are deleted, substituted, or added means that a number of amino acids (preferably 10 or fewer, more preferably 7 or fewer, and even more preferably 6 or fewer) that can be deleted, substituted, or added by methods such as site-directed mutagenesis or other mutant peptide production methods are deleted, substituted, or added.

[0054] Furthermore, examples of substantially identical amino acid sequences include amino acid sequences that have an identity of 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 92% or more, particularly preferably 95% or more, and most preferably 99% or more, with amino acid sequences obtainable from known databases.

[0055] Examples of R-spongins include the R-spongin family, which consists of R-spongin 1, R-spongin 2, R-spongin 3, and R-spongin 4. The R-spongin family are secreted proteins and are known to be involved in the activation and regulation of the Wnt signaling pathway. Multiple types of R-spongins may be used in combination in the culture medium for epithelial cell-derived organoids. Furthermore, R-spongin fragments may be used as long as they possess R-spongin activity, and may contain amino acid sequences other than those of R-spongin.

[0056] The concentration of Wnt protein in the culture medium for epithelial cell-derived organoids is preferably 50 ng / mL or higher, more preferably 100 ng / mL to 10 μg / mL, even more preferably 200 ng / mL to 1 μg / mL, and particularly preferably 300 ng / mL to 1 μg / mL.

[0057] Examples of GSK-3β inhibitors include CHIR-99021 (CAS number: 252917-06-9), CHIR-98014 (CAS number: 252935-94-7), lithium (Sigma), Kaempaulon (CAS number: 142273-20-9), 6-bromoindilbine-30-acetoxime, SB216763 (CAS number: 280744-09-4), SB415286 (CAS number: 264218-23-7), FRAT family members that inhibit the interaction between GSK-3 and axin, and FRAT-derived peptides.

[0058] Afamin is a glycoprotein belonging to the albumin family and is known to be present in blood or body fluids. Serum contains afamin derived from the animal from which the serum was collected. Since serum contains impurities other than afamin, it is preferable that the cell culture medium contains afamin alone and does not contain serum.

[0059] The origin of afamin is not particularly limited, and afamin from various organisms can be used. Among these, mammalian afamin is preferred. The amino acid sequences of major mammalian afamins and the base sequences of the genes encoding them can be obtained from known databases such as GenBank. For example, in GenBank, the amino acid sequence of human afamin is registered with accession number AAA21612, and the base sequence of the gene encoding it is registered with accession number L32140, while the amino acid sequence of bovine afamin is registered with accession number DAA28569, and the base sequence of the gene encoding it is registered with accession number GJ060968.

[0060] The afamin contained in the cell culture medium may be naturally occurring afamin found in serum or the like, purified by a known method, or it may be genetically modified afamin.

[0061] Wnt proteins are highly hydrophobic because certain serine residues are modified with fatty acids (palmitoleic acid). Therefore, Wnt proteins are known to be very difficult to purify and store in aqueous solutions due to their tendency to aggregate or denature. On the other hand, this fatty acid modification of specific serine residues is essential for the physiological activity of Wnt proteins and has been reported to be involved in binding to Frizzled receptor family members. Furthermore, there are findings suggesting that in aqueous solutions, Wnt proteins can bind to afamin in a one-to-one ratio, forming a complex and becoming solubilized while maintaining high physiological activity.

[0062] Therefore, the Wnt protein-afamine complex may be produced by culturing cells that express both Wnt protein and afamin, or by co-culturing Wnt protein-expressing cells and afamin-expressing cells.

[0063] The concentration of afamin in the culture medium for epithelial cell-derived organoids is not particularly limited, but is preferably 50 ng / mL to 10 μg / mL, more preferably 100 ng / mL to 1 μg / mL, and even more preferably 300 μg / mL to 1 μg / mL.

[0064] The culture medium for epithelial cell-derived organoids may further contain a Rock (Rho-kinase) inhibitor. Examples of Rock inhibitors include Y-27632 ((R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate), Fasudil (HA1077) (5-(1,4-diazepan-1-ylsulfonyl)isoquinoline), and H-1152 ((S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride). When using Y-27632 as the Rock inhibitor, it is preferable to add it during the first two days of culturing epithelial cells dispersed in single cells. The concentration of Y-27632 in the culture medium for epithelial cell-derived organoids is preferably about 10 μM.

[0065] The culture medium for epithelial cell-derived organoids may further contain gastrin (or a suitable substitute such as Leu15-gastrin). The concentration of gastrin (or a suitable substitute) in the culture medium for epithelial cell-derived organoids is preferably 1 ng / mL to 10 μg / mL, more preferably 1 ng / mL to 1 μg / mL, and even more preferably 5 to 100 ng / mL.

[0066] The culture medium for epithelial cell-derived organoids may further contain at least one amino acid. Examples of amino acids include L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and combinations thereof. The concentration of L-glutamine in the culture medium for epithelial cell-derived organoids is preferably 0.05 to 1 g / L, and more preferably 0.1 to 0.75 g / L. The other amino acids in the culture medium for epithelial cell-derived organoids are preferably 0.001 to 1 g / L, and more preferably 0.01 to 0.15 g / L.

[0067] The culture medium for epithelial cell-derived organoids may further contain at least one vitamin. Examples of vitamins include thiamine (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), calcium D-pantothenate (vitamin B5), pyridoxal / pyridoxamine / pyridoxine (vitamin B6), folic acid (vitamin B9), cyanocobalamin (vitamin B12), ascorbic acid (vitamin C), calciferol (vitamin D2), DL-α-tocopherol (vitamin E), biotin (vitamin H), and menadione (vitamin K).

[0068] Culture media for epithelial cell-derived organoids may further contain at least one inorganic salt. Inorganic salts have the function of helping to maintain the osmotic equilibrium of cells and regulating membrane potential. Specific examples of inorganic salts include salts of calcium, copper, iron, magnesium, potassium, sodium, and zinc. Salts are usually used in the form of chlorides, phosphates, sulfates, nitrates, and bicarbonates. More specific examples of salts include CaCl2, CuSO4-5H2O, Fe(NO3)-9H2O, FeSO4-7H2O, MgCl, MgSO4, KCl, NaHCO3, NaCl, Na2HPO4, Na2HPO4-H2O, and ZnSO4-7H2O.

[0069] The culture medium for epithelial cell-derived organoids may further contain at least one sugar that can serve as a carbon energy source. Examples of sugars include glucose, galactose, maltose, and fructose. Among these, glucose is preferred, and D-glucose (dextrose) is particularly preferred. The concentration of sugar in the culture medium for epithelial cell-derived organoids is preferably 1 to 10 g / L.

[0070] The culture medium for epithelial cell-derived organoids may further contain at least one trace element. Examples of trace elements include barium, bromium, cobalt, iodine, manganese, chromium, copper, nickel, selenium, vanadium, titanium, germanium, molybdenum, silicon, iron, fluorine, silver, rubidium, tin, zirconium, cadmium, zinc, aluminum, and their ions.

[0071] The culture medium for epithelial cell-derived organoids may further contain at least one additional agent. Examples of agents include nutrients or growth factors that have been reported to improve stem cell culture, such as cholesterol, transferrin, albumin, insulin, progesterone, putrescine, and selenite.

[0072] [Method for producing total RNA] In one embodiment, the present invention provides a method for producing total RNA from animal cells co-cultured with microorganisms, wherein the ratio of the content of 28S rRNA to the content of 18S rRNA (28S rRNA / 18S rRNA) is 2 or more, or the absorbance at a wavelength of 260 nm (A 260 ) and the absorbance at a wavelength of 280 nm (A 280 ) ratio (A 260 / A 280 ) is 2 or more, the method comprising: co-culturing microorganisms and animal cells in a medium containing 15 mM or more of a buffer having a pKa of 6.0 to 9.0 at 20 °C; and preparing total RNA from the animal cells.

[0073] As will be described later in the examples, the inventors have found that high-purity total RNA cannot be recovered from animal cells co-cultured with microorganisms using a conventional medium for organoid culture. In contrast, the method of the present embodiment can produce high-purity total RNA from animal cells co-cultured with microorganisms.

[0074] Generally, high-purity total RNA has a ratio of the content of 28S rRNA to the content of 18S rRNA (content of 28S rRNA / content of 18S rRNA) of 2 or more, and the upper limit is considered to be infinite. Also, generally, high-purity total RNA has a ratio of the absorbance at a wavelength of 260 nm (A 260 ) and the absorbance at a wavelength of 280 nm (A 280 ) (A 260 / A 280 ) of 2 or more, and the upper limit is considered to be infinite.

[0075] The ratio of the content of 28S rRNA to the content of 18S rRNA in total RNA can be determined, for example, by electrophoresing the total RNA, measuring the intensities of the bands of 28S rRNA and 18S rRNA, and calculating their ratio. The electrophoresis may be performed by gel electrophoresis or using a microchip electrophoresis apparatus.

[0076] The ratio of the absorbance at a wavelength of 260 nm to the absorbance at a wavelength of 280 nm can be determined by measuring the absorbance of the total RNA using an absorbance altimeter or spectrophotometer and calculating the ratio between the two.

[0077] Total RNA can be prepared from animal cells using commercially available kits. Specific examples of such kits include the RNeasy kit (Qiagen), RNA rapid extraction solution (Thermo Fisher Scientific), and TRIzol Reagent (Thermo Fisher Scientific).

[0078] In the manufacturing method of this embodiment, the microorganisms, animal cells, buffer with a pKa of 6.0 to 9.0 at 20°C, and culture medium are the same as those described above.

[0079] [Other embodiments] In one embodiment, the present invention provides a co-culture comprising a culture medium, microorganisms, and animal cells, containing 15 mM or more of a buffering agent having a pKa of 6.0 to 9.0 at 20°C.

[0080] In the co-culture of this embodiment, the microorganisms, animal cells, buffer with a pKa of 6.0 to 9.0 at 20°C, and culture medium are the same as those described above.

[0081] The co-culture of this embodiment may be housed in the culture vessel shown in Figure 1. As described above, the culture vessel shown in Figure 1 comprises an upper vessel and a lower vessel, and at least a portion of the bottom surface of the upper vessel is composed of a membrane that allows at least a portion of the components of the culture medium to pass through but not the cells. Epithelial cells are cultured inside the upper vessel, and when the epithelial cells reach a confluent state, the upper vessel becomes isolated, and the inside of the upper vessel can be maintained under anaerobic conditions. [Examples]

[0082] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0083] [Experimental Example 1] (Examination of RNA quality 1) Human normal colon epithelial organoid-derived cells were co-cultured with Bifidobacterium longum, a type of Bifidobacterium, using the culture vessel shown in Figure 1. After co-culture, total RNA was extracted from the human cells, and the RNA quality was evaluated.

[0084] First, human normal colon epithelial organoids cultured in 3D were broken down into single cells using TrypLE Express (Thermo Fisher Scientific). Next, the obtained single cells were suspended in cell culture medium and divided into 5 × 10⁻⁶ cells. 5 The cells were seeded into the upper container and cultured for 5 days in an incubator under normal oxygen conditions at 37°C. As a result, the monolayer cultured epithelial cells became confluent, and the upper container became isolated.

[0085] Next, add 1 × 10 to the culture medium (upper layer medium) in the upper container. 7 Bifidobacterium longum CFUs (Colony Forming Units) were added and co-cultured with epithelial cells. The volume of the upper culture medium was 200 μL. Animal cell culture media containing HEPES at concentrations of 10 mM, 20 mM, 40 mM, 60 mM, 80 mM, and 100 mM were used as both the upper culture medium and the lower culture medium in the lower container. The volume of the lower culture medium was 600 μL. The animal cell culture medium containing 10 mM HEPES is a standard organoid culture medium.

[0086] The animal cell culture medium was Advanced DMEM / F-12 medium (Thermo Fisher Scientific) supplemented with conditioned medium for W-Wnt3a / HEK cells cultured in serum-containing medium containing 1 μg / mL human recombinant R-spongin 1 (R&D Systems), 100 ng / mL Noggin (Peprotec), 500 nM A83-01 (Tocris), and 300 ng / mL Wnt3a, as well as 100 ng / mL IGF1 (Biolegend), 50 ng / mL FGF2 (Peprotec), 50 ng / mL mouse recombinant EGF (Thermo Fisher Scientific), and 10 μM Y-27632 (Fujifilm Wako Pure Chemical Industries). The upper container was maintained under anaerobic conditions, while the lower container was maintained under normal oxygen-containing conditions.

[0087] Next, epithelial cells were harvested 24 hours after the start of co-culture, and total RNA was extracted. A commercially available kit (RNeasy Mini Kit, Qiagen) was used for total RNA extraction. Subsequently, the quality of the total RNA was confirmed by RNA analysis microchip electrophoresis.

[0088] Figure 2 shows images of the results of RNA analysis microchip electrophoresis. In Figure 2, "Bifidobacterium" represents Bifidobacterium longum, and "vehicle" represents a culture medium that does not contain Bifidobacterium longum.

[0089] In Figure 2, lane 1 shows the total RNA results when animal cell medium containing 10 mM HEPES was used and Bifidobacterium longum was not added. Lane 2 shows the total RNA results when animal cell medium containing 10 mM HEPES was used and Bifidobacterium longum was added. Lane 3 shows the total RNA results when animal cell medium containing 20 mM HEPES was used and Bifidobacterium longum was added. Lane 4 shows the total RNA results when animal cell medium containing 40 mM HEPES was used and Bifidobacterium longum was added. Lane 5 shows the total RNA results when animal cell medium containing 60 mM HEPES was used and Bifidobacterium longum was added. Lane 6 shows the total RNA results when animal cell medium containing 80 mM HEPES was used and Bifidobacterium longum was added. Lane 7 shows the total RNA results when using animal cell medium containing 100 mM HEPES without the addition of Bifidobacterium longum. Lane 8 shows the total RNA results when using animal cell medium containing 100 mM HEPES with the addition of Bifidobacterium longum.

[0090] Figure 3 is a graph showing the results of quantifying the band intensity for lanes 1-4 in Figure 2. Figure 4 is a graph showing the results of quantifying the band intensity for lanes 5-8 in Figure 2. Figure 5 is a graph showing the results of calculating the ratio of 28S rRNA content to 18S rRNA content (28S rRNA content / 18S rRNA content) of total RNA in lanes 1-8 of Figure 2, based on the results of Figures 3 and 4.

[0091] Figure 6 shows the absorbance (A) at a wavelength of 260 nm for all RNA in lanes 1-8 of Figure 2. 260 ) and absorbance at a wavelength of 280 nm (A 280 ) ratio (A 260 / A 280 This graph shows the results of calculating ).

[0092] Figure 7 is a graph showing the results of measuring the total RNA mass extracted from epithelial cells cultured in monolayers under each condition.

[0093] As a result, the results from lane 1 confirmed that high-quality RNA could be extracted from human cells (control) cultured in standard organoid culture medium without co-culturing with Bifidobacterium longum.

[0094] However, the results from lane 2 showed that RNA extracted from human cells co-cultured with Bifidobacterium longum in standard organoid culture medium was degraded.

[0095] Furthermore, results from lanes 3-6 and 8 demonstrated that high-quality RNA can be extracted from human cells co-cultured with Bifidobacterium longum in animal cell culture medium containing 20 mM-100 mM HEPES.

[0096] Furthermore, as shown in Figure 7, RNA extracted from human cells co-cultured with Bifidobacterium longum in standard organoid culture medium tended to be lower compared to other conditions. Additionally, RNA extracted from human cells co-cultured with Bifidobacterium longum in animal cell medium containing 20 mM to 100 mM HEPES was found to be at a similar level to the control.

[0097] [Experimental Example 2] (Investigation of the effects of HEPES on microorganisms) In Experimental Example 1, human normal colon epithelial organoid-derived cells and Bifidobacterium longum were co-cultured for 24 hours under each condition. After that, the Bifidobacterium longum was harvested and seeded on anaerobic bacterial agar medium, and the colony-forming units were measured.

[0098] Figure 8 shows photographs of Bifidobacterium longum colonies co-cultured with human cells in animal cell culture media containing various concentrations of HEPES.

[0099] Figure 9 is a graph showing the colony-forming units calculated based on the results in Figure 8. The results showed that Bifidobacterium longum grew without problems at all HEPES concentrations from 10 mM to 100 mM.

[0100] The results from Experimental Examples 1 and 2 demonstrate that the animal cell culture medium of the present invention is useful for stably co-culturing microorganisms and animal cells.

[0101] [Experimental Example 3] (Examination of RNA quality 2) Human normal colon epithelial organoid-derived cells were co-cultured with Bifidobacterium longum, a type of Bifidobacterium, using the culture vessel shown in Figure 1. After co-culture, total RNA was extracted from the human cells, and the RNA quality was evaluated.

[0102] First, human normal colon epithelial organoids cultured in 3D were broken down into single cells using TrypLE Express (Thermo Fisher Scientific). Next, the obtained single cells were suspended in cell culture medium and divided into 5 × 10⁻⁶ cells. 5 The cells were seeded into the upper container and cultured for 5 days in an incubator under normal oxygen conditions at 37°C. As a result, the monolayer cultured epithelial cells became confluent, and the upper container became isolated.

[0103] Next, add 1 × 10 to the culture medium (upper layer medium) in the upper container. 7 Bifidobacterium longum CFUs (Colony Forming Units) were added and co-cultured with epithelial cells. The volume of the upper culture medium was 200 μL. Animal cell culture media containing MES at concentrations of 10 mM, 20 mM, 40 mM, 60 mM, 80 mM, and 100 mM were used as both the upper culture medium and the lower culture medium in the lower container. The volume of the lower culture medium was 600 μL.

[0104] The animal cell culture medium was Advanced DMEM / F-12 medium (Thermo Fisher Scientific) supplemented with conditioned medium for W-Wnt3a / HEK cells cultured in serum-containing medium containing 1 μg / mL human recombinant R-spongin 1 (R&D Systems), 100 ng / mL Noggin (Peprotec), 500 nM A83-01 (Tocris), and 300 ng / mL Wnt3a, as well as 100 ng / mL IGF1 (Biolegend), 50 ng / mL FGF2 (Peprotec), 50 ng / mL mouse recombinant EGF (Thermo Fisher Scientific), and 10 μM Y-27632 (Fujifilm Wako Pure Chemical Industries). The upper container was maintained under anaerobic conditions, while the lower container was maintained under normal oxygen-containing conditions.

[0105] Next, epithelial cells were harvested 24 hours after the start of co-culture, and total RNA was extracted. A commercially available kit (RNeasy Mini Kit, Qiagen) was used for total RNA extraction. Subsequently, the quality of the total RNA was confirmed by RNA analysis microchip electrophoresis.

[0106] Figure 10 shows images of the results of RNA analysis microchip electrophoresis. In Figure 10, "Bifidobacterium" represents Bifidobacterium longum, and "vehicle" represents a culture medium that does not contain Bifidobacterium longum.

[0107] In Figure 10, lane 1 shows the total RNA results when animal cell medium containing 10 mM MES was used and Bifidobacterium longum was not added. Lane 2 shows the total RNA results when animal cell medium containing 20 mM MES was used and Bifidobacterium longum was not added. Lane 3 shows the total RNA results when animal cell medium containing 40 mM MES was used and Bifidobacterium longum was not added. Lane 4 shows the total RNA results when animal cell medium containing 100 mM MES was used and Bifidobacterium longum was not added. Lane 5 shows the total RNA results when animal cell medium containing 10 mM MES was used and Bifidobacterium longum was added. Lane 6 shows the total RNA results when animal cell medium containing 20 mM MES was used and Bifidobacterium longum was added. Lane 7 shows the total RNA results when animal cell medium containing 40 mM MES was used and Bifidobacterium longum was added. Lane 8 shows the total RNA results when animal cell medium containing 100 mM MES was used and Bifidobacterium longum was added.

[0108] Figure 11 is a graph showing the results of quantifying the band intensity for lanes 1-4 in Figure 10. Figure 12 is a graph showing the results of quantifying the band intensity for lanes 5-8 in Figure 10. Figure 13 is a graph showing the results of calculating the ratio of 28S rRNA content to 18S rRNA content (28S rRNA content / 18S rRNA content) of total RNA in lanes 1-8 of Figure 10, based on the results of Figures 11 and 12.

[0109] Figure 14 shows the absorbance (A) at a wavelength of 260 nm for all RNA in lanes 1-8 of Figure 10. 260 ) and absorbance at a wavelength of 280 nm (A 280 ) ratio (A 260 / A 280 This graph shows the results of calculating ).

[0110] Figure 15 is a graph showing the results of measuring the concentration of total RNA extracted from epithelial cells cultured in monolayers under each condition. The total volume of extracted total RNA was 50 μL.

[0111] As a result, the findings from lanes 1-4 confirmed that high-quality RNA could be extracted from human cells that were not co-cultured with Bifidobacterium longum.

[0112] However, the results from lane 5 showed that RNA extracted from human cells co-cultured with Bifidobacterium longum in organoid culture medium containing 10 mM MES was degraded.

[0113] Furthermore, results from lanes 6-8 demonstrated that high-quality RNA can be extracted from human cells co-cultured with Bifidobacterium longum in animal cell culture medium containing 20 mM to 100 mM MES.

[0114] Furthermore, as shown in Figure 15, no significant difference was observed in the amount of RNA extracted from human cells co-cultured with Bifidobacterium longum under any of the conditions.

[0115] [Experimental Example 4] (Investigation of the effects of MES on microorganisms) In Experimental Example 3, human normal colon epithelial organoid-derived cells and Bifidobacterium longum were co-cultured for 24 hours under each condition. After that, the Bifidobacterium longum was harvested and seeded on anaerobic bacterial culture agar medium, and the colony-forming units were measured.

[0116] Figure 16 shows photographs of Bifidobacterium longum colonies co-cultured with human cells in animal cell culture media containing various concentrations of MES.

[0117] Figure 17 is a graph showing the colony-forming units calculated based on the results in Figure 16. The results showed that Bifidobacterium longum grew without problems at all MES concentrations from 10 mM to 100 mM.

[0118] The results of Experimental Examples 3 and 4 further support the usefulness of the animal cell culture medium of the present invention for the stable co-culture of microorganisms and animal cells. [Industrial applicability]

[0119] According to the present invention, a technology for stably co-culturing microorganisms and animal cells can be provided. According to the present invention, highly pure total RNA can be recovered from animal cells co-cultivated with microorganisms.

Claims

1. A culture medium for co-culturing microorganisms and animal cells, containing 15 mM or more of a buffering agent with a pKa of 6.0 to 9.0 at 20°C.

2. The buffering agent is 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris), N-(2-acetamide)iminodiacetic acid (ADA), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), N-(2-acetamide)-2-aminoethanesulfonic acid (ACES), 2-hydroxy-3-morpholinopropanesulfonic acid (MOPSO), N-[tris(hydroxymethyl)methyl]glycine (Tricine), N,N-bis(2-hydroxyethyl)glycine (Bicine), 2-[N,N-bis(2-hydroxyethyl)amino]-1-ethane The culture medium according to claim 1, comprising sulfonic acid (BES), 3-morpholinopropane-1-sulfonic acid (MOPS), N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), 3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid (TAPSO), piperazine-1,4-bis(2-hydroxypropanesulfonic acid) (POPSO), 4-(2-hydroxyethyl)piperazine-1-(2-hydroxypropane-3-sulfonic acid) (HEPPSO), 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS), tricine, bicine, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), glycinamide, or acetamidoglycine.

3. The culture medium according to claim 1 or 2, wherein the animal cells are epithelial cells.

4. From animal cells co-cultured with microorganisms, the ratio of 28S rRNA content to 18S rRNA content (28S rRNA / 18S rRNA) is 2 or greater, or the absorbance at a wavelength of 260 nm (A ２６０ ) and absorbance at a wavelength of 280 nm (A ２８０ ) ratio (A ２６０ / A ２８０ A method for producing total RNA in which ) is 2 or more, A step of co-culturing microorganisms and animal cells in a culture medium containing 15 mM or more of a buffering agent having a pKa of 6.0 to 9.0 at 20°C, A step of preparing total RNA from the aforementioned animal cells, A manufacturing method that includes this.

Images