Application of prostaglandin E2 in maintaining state of fetal-like intestine stem cells

Abstract

The invention relates to a special intestinal organ culture method, and particularly emphasizes the core effect of prostaglandin E2 in maintaining the state of fetal-like intestinal stem cells. A culture medium used in the method consists of a basic culture medium, a serum substitute, antibiotics, glutamate / ester, an HEPES buffer solution, EGF (Epidermal Growth Factor), Noggin, R-spondin and prostaglandin E2. Wherein the prostaglandin E2 is used as a key component, the expression of related genes of adult intestinal stem cells is remarkably inhibited by finely regulating a Wnt / beta-catenin signal channel, the expression of related genes of undifferentiated fetal-like intestinal stem cells is promoted, the precious fetal-like state of the cells is maintained, and the cells can stably grow and pass. The culture medium provided by the invention not only provides an efficient fetal-like intestine stem cell culture scheme, but also opens up a new way for stem cell therapy through the unique effect of prostaglandin E2, and provides new possibility for treatment of intestinal diseases and tissue engineering.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, specifically to the development of a culture medium and its application, covering a special culture medium and its use in biomedical research and treatment, and more specifically to the application of prostaglandin E2 in maintaining a fetal-like intestinal stem cell state. Background Art

[0002] The intestine is an integral part of the human digestive system, responsible for digesting food, absorbing nutrients, and maintaining the overall health of the body. It not only provides nutrients and energy required for life, but also plays an important role in immune defense. The integrity of the intestine is essential for maintaining health, and the renewal and repair of the intestinal epithelium depends on the presence of intestinal stem cells.

[0003] Intestinal stem cells are a type of cell with the ability to self-renew and differentiate, and can generate various intestinal epithelial cells to maintain the normal functioning of the intestine. These stem cells are divided into active and quiescent types, the former proliferating rapidly when needed, while the latter remain in a quiescent state. In recent years, researchers have discovered a new type of intestinal stem cell - fetal-like intestinal stem cells, which not only have good proliferation ability, but can also differentiate into a variety of intestinal epithelial cell types.

[0004] The study of fetal-like intestinal stem cells is of great significance. They provide new hope for regenerative medicine and may play a role in the treatment of intestinal diseases, tissue repair, and drug screening. For example, the treatment of intestinal inflammatory diseases such as Crohn's disease and ulcerative colitis may benefit from these cells because they can help restore damaged intestinal epithelium. In addition, fetal-like intestinal stem cells can be used to develop new drug screening platforms to improve the efficiency of drug development. In short, in-depth research on the intestine and its stem cells can not only promote the development of basic science, but also lay the foundation for future clinical applications.

[0005] Currently, the scientific community has not yet developed a mature technology to obtain and culture fetal-like intestinal stem cells on a large scale and maintain their state in the process so that they can grow and pass down normally. This field is still in the exploratory stage and further research and innovation are needed to overcome existing challenges. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides the use of prostaglandin E2 in maintaining the state of fetal-like intestinal stem cells. The culture medium provided by the present invention includes basal culture medium, serum replacement, antibiotics, glutamate / ester, HEPES buffer, EGF, Noggin, R-spondin and prostaglandin E2, which can be used to efficiently obtain and culture intestinal organoids rich in fetal-like intestinal stem cells. Among them, prostaglandin E2, as a key component, significantly reduces the expression of genes related to differentiated adult intestinal stem cells by regulating the Wnt / β-catenin signaling pathway, and significantly increases the expression of genes in undifferentiated fetal-like intestinal stem cells. The intestinal crypts wrapped in matrix gel are placed in the above culture medium for culture, and fetal-like intestinal stem cells can be obtained; the fetal-like intestinal stem cells are placed in the above culture medium for continued culture, and the state can be stably maintained and normally passaged.

[0007] The first object of the present invention is to provide the use of prostaglandin E2 in maintaining the fetal-like intestinal stem cell state;

[0008] The application uses a culture medium to convert intestinal epithelial cells into fetal-like intestinal stem cells in vitro and maintain the fetal-like intestinal stem cell state;

[0009] The culture medium comprises a basal culture medium, a serum replacement, antibiotics, glutamate / ester, a HEPES buffer, epidermal growth factor EGF, Noggin, R-spondin and prostaglandin E2;

[0010] The concentration of the prostaglandin E2 in the culture medium is 3-7 μg / mL.

[0011] Among them, prostaglandin E2 (P for short) is a type of prostaglandin, which has multiple functions such as regulating blood pressure, inflammatory response, and promoting cell proliferation and differentiation.

[0012] Furthermore, the intestinal epithelial cells are derived from the small intestine.

[0013] Furthermore, the intestinal epithelial cells are mouse intestinal epithelial cells.

[0014] Furthermore, the basal culture medium is DMEM / F12 complete culture medium.

[0015] Furthermore, the serum substitutes are N2 and B27.

[0016] Furthermore, the antibiotics are penicillin and streptomycin.

[0017] Furthermore, the glutamate / ester is Glutamax, and the concentration of Glutamax is 2mM.

[0018] Furthermore, the concentration of the HEPES buffer is 1M.

[0019] Furthermore, the concentration of the epidermal growth factor EGF is 30-70 ng / mL, the concentration of Noggin is 50-150 ng / mL, and the concentration of R-spondin is 500-1000 ng / mL.

[0020] Preferably, the concentration of the epidermal growth factor EGF is 50 ng / mL.

[0021] Preferably, the concentration of Noggin is 100 ng / mL.

[0022] Preferably, the concentration of R-spondin is 1000 ng / mL.

[0023] Preferably, the concentration of prostaglandin E2 is 5 μg / mL.

[0024] The second object of the present invention is to provide a culture medium for maintaining a fetal-like intestinal stem cell state, comprising a basal culture medium, a serum replacement, antibiotics, glutamate / ester, a HEPES buffer, epidermal growth factor EGF, Noggin, R-spondin and prostaglandin E2;

[0025] The basal culture medium is DMEM / F12 complete culture medium;

[0026] The serum replacements are N2 and B27;

[0027] The glutamate / ester is Glutamax, and the concentration of Glutamax is 2mM;

[0028] The concentration of the HEPES buffer is 1M;

[0029] The concentration of the epidermal growth factor EGF is 30-70 ng / mL, the concentration of the Noggin is 50-150 ng / mL, and the concentration of the R-spondin is 500-1000 ng / mL;

[0030] The concentration of prostaglandin E2 is 3-7 μg / mL.

[0031] According to an embodiment of the present invention, the culture medium is capable of dedifferentiating small intestinal organoids cultured in vitro into intestinal organoids rich in fetal-like intestinal stem cells, and the latter are capable of normal growth, maintaining a stable state and being passaged.

[0032] Preferably, the concentration of the epidermal growth factor EGF is 50 ng / mL.

[0033] Preferably, the concentration of Noggin is 100 ng / mL.

[0034] Preferably, the concentration of R-spondin is 1000 ng / mL.

[0035] Preferably, the concentration of prostaglandin E2 is 5 μg / mL.

[0036] The third object of the present invention is to provide a method for obtaining fetal-like intestinal stem cells in vitro, wherein the intestinal crypts wrapped in matrix gel are placed in the above culture medium for culture.

[0037] Further, the intestinal crypts are isolated from the small intestine.

[0038] Furthermore, the culture is carried out under sterile conditions, 5% CO2 and 37°C.

[0039] In one embodiment of the present invention, the culturing time is 48 hours.

[0040] In one embodiment of the present invention, the method for isolating intestinal crypts comprises the following steps:

[0041] (1) Place the small intestine in pre-cooled sterile DPBS buffer, rinse it clean, and process the small intestine into small intestinal segments;

[0042] (2) Place the intestinal segment in pre-cooled DPBS buffer, place it on ice, and gently shake it on a shaker at 20 rpm for 5 minutes for a second wash;

[0043] (3) Discard the DPBS buffer, add pre-cooled DPBS buffer containing 2 mM EDTA, and continue shaking on ice at 20 rpm for 30 min;

[0044] (4) Discard the DPBS buffer containing 2 mM EDTA and add pre-cooled DPBS buffer containing 55 mM sorbitol and 43 mM sucrose to rapidly and vigorously remove the crypts from the intestinal tube;

[0045] (5) Use a sterile cell strainer to filter the buffer. The crypts will remain in the filtered buffer.

[0046] (6) The filtered buffer was centrifuged at 900 rpm for 5 minutes at 4°C to collect pure small intestinal crypt cells.

[0047] The fourth object of the present invention is to provide a method for culturing fetal-like intestinal stem cells, wherein the fetal-like intestinal stem cells encapsulated in matrix gel are placed in the above culture medium for culturing, and the fetal-like intestinal stem cells are obtained by the above method.

[0048] Furthermore, the culture is carried out under sterile conditions, 5% CO2 and 37°C.

[0049] Beneficial effects of the present invention:

[0050] (1) Prostaglandin E2 is a small molecule compound that does not cause changes in inherent biological properties, including genomic information. The present invention adds prostaglandin E2 to the culture medium to maintain the state of fetal-like intestinal stem cells, significantly reduces the expression of genes related to differentiated adult intestinal stem cells by regulating the Wnt / β-catenin signaling pathway, and significantly increases the expression of genes in undifferentiated fetal-like intestinal stem cells, thereby ensuring that the cells remain in a fetal-like state.

[0051] (2) Using the culture medium provided by the present invention to culture small intestinal organoids in vitro can increase the expression of the fetal-like intestinal stem cell marker gene Sca1, reduce the expression of adult intestinal stem cell marker genes Lgr5, Hopx and Lrig1, inhibit the expression of genes related to the Wnt signaling pathway, and induce the transformation of ordinary intestinal organoids into intestinal organoids rich in fetal-like intestinal stem cells, thereby solving the problem of the difficulty in obtaining fetal-like intestinal stem cells.

[0052] (3) The method for obtaining and / or culturing fetal-like intestinal stem cells in vitro provided by the present invention not only simplifies the culture process of fetal-like intestinal stem cells and enables small intestinal epithelial cells to differentiate into fetal-like intestinal stem cells under in vitro conditions, but also has the advantages of an original and mature culture system: it contains growth factors, thereby ensuring that intestinal organoids rich in fetal-like intestinal stem cells grow well and maintain their normal state. This provides strong support for the promotion of the in vitro culture system and acquisition method of fetal-like intestinal stem cells, is conducive to the large-scale in vitro expansion and stable passaging of fetal-like intestinal stem cells, and provides a valuable platform for in-depth research on fetal-like intestinal stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0054] Figure 1 is the chemical structural formula of the small molecule drug prostaglandin E2 of the present invention;

[0055] Figure 2 The difference in growth status of intestinal organoids under different culture conditions in Example 1 of the present invention, wherein A is the morphological appearance of intestinal organoids cultured in the ENR and ENRP systems, and B is the percentage of the number of spherical organoids and budding organoids cultured in the ENR and ENRP systems;

[0056] Figure 3The expression level results of the fetal-like intestinal stem cell marker gene Sca1 in intestinal organoids under different culture conditions in Example 2 of the present invention, wherein A is the expression difference of the Sca1 gene in different culture systems, B is the GSEA enrichment analysis result, and C is a heat map showing the expression of fetal-like intestinal stem cell-related genes in the transcriptome data;

[0057] Figure 4 The expression level results of the active adult intestinal stem cell marker gene Lgr5 in intestinal organoids under different culture conditions in Example 2 of the present invention, wherein A is the expression difference of the Lgr5 gene in different culture systems, B is the GSEA enrichment analysis result, and C is a heat map showing the expression of the active adult intestinal stem cell marker gene in the transcriptome data;

[0058] Figure 5 The expression levels of quiescent adult intestinal stem cell marker genes (Hopx and Lrig1) in intestinal organoids under different culture conditions in Example 2 of the present invention;

[0059] Figure 6 These are the expression level results of genes related to the Wnt signaling pathway in intestinal organoids under different culture conditions in Example 2 of the present invention, wherein A is the expression difference of the Axin2 gene in different culture systems, B is the GSEA enrichment analysis result, and C is a heat map showing the expression of genes related to the Wnt signaling pathway in the transcriptome data. DETAILED DESCRIPTION

[0060] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0061] The reagents used in the following examples are shown in Table 1:

[0062] Table 1 Reagents and sources

[0063] Reagents source Model / Concentration Advanced DMEM / F12 Gibco Corporation (USA) N2-max supplement R&D Systems (USA) #AR009 B27supplement Gibco Corporation (USA) penicillin Invitrogen Corporation (USA) 100units / mL Streptomycin Invitrogen Corporation (USA) 100 μg / mL Glutamax Thermo Fisher Corporation (USA) HEPES buffer Sigma Corporation (USA)

[0064] The composition of the culture medium involved in the following examples is shown in Table 2:

[0065] Table 2 Culture medium composition

[0066]

[0067] Example 1: Acquisition and culture of intestinal organoids rich in fetal-like intestinal stem cells

[0068] 1. Methods for obtaining intestinal organoids rich in fetal-like intestinal stem cells

[0069] (1) Take a 6-8 week old mouse, use dry sterile scissors and straight forceps to remove the small intestine of about 10 cm in length, and quickly put it into pre-cooled sterile DPBS buffer;

[0070] (2) Use cold DPBS solution to flush the small intestine to prevent digestive residues from remaining in the intestine. After flushing, place the small intestine back into the pre-cooled sterile DPBS buffer;

[0071] (3) Cut the small intestine into small segments of about 0.5 cm in length, transfer them to a 15 mL centrifuge tube, add 5 mL of sterile pre-cooled DPBS buffer, place it on ice, and gently shake it on a shaker at 20 rpm for 5 minutes for a second wash;

[0072] (4) After washing, discard the DPBS buffer and add pre-cooled DPBS buffer containing 2 mM EDTA to the centrifuge tube, and continue shaking on ice at 20 rpm for 30 minutes;

[0073] (5) Discard the DPBS buffer containing EDTA, add pre-cooled DPBS buffer containing 55 mM sorbitol and 43 mM sucrose, and shake rapidly and vigorously to remove the crypts from the intestinal tube;

[0074] (6) Use a sterile cell sieve with a pore size of 70 μm (FALCON, USA) to filter the buffer. Since the crypts are small in size, they will remain in the filtered buffer. Place the filtered buffer at 4°C and centrifuge at 900 rpm for 5 minutes to collect pure small intestinal crypt cells;

[0075] (7) Every 500 isolated crypt cells were taken as a group and mixed evenly with 50 μL of pre-cooled matrix gel and PBS buffer solution (volume ratio of 1:1) and plated in a single well of a 24-well cell culture plate. The culture plate was placed in a 37°C constant temperature cell culture incubator for 30 minutes to 1 hour. After the matrix gel solidified into a solid, the ENR system was carefully added near the well wall for culture.

[0076] 2. Cultivation Method of Intestinal Organoids Enriched with Fetal-like Intestinal Stem Cells

[0077] (1) After about 4-5 days of in vitro culture of intestinal organoids, dead cell fragments will accumulate in the lumen of the intestinal organoids. In order to eliminate the possible impact of cell fragments on the intestinal organoids, subculture should be performed in a timely manner to maintain the health of the intestinal organoids;

[0078] (2) Take the 24-well plate out of the cell culture incubator and place it on ice. Remove the original culture medium and wash it with sterile pre-cooled DPBS buffer. After washing, discard the DPBS buffer.

[0079] (3) Add 1 mL of cold DPBS buffer to each well, scrape the matrix gel and DPBS buffer and gently blow until they are fragmented, and rinse the well with a small amount of DPBS buffer until no matrix gel remains on the cell plate;

[0080] (4) Use a 1 mL syringe to absorb the broken intestinal organoid matrix gel suspension and add it to a 15 mL centrifuge tube placed on ice. Squeeze and separate the intestinal organoid "buds" (intestinal stem cells) from the intestinal organoid cell bodies (epithelial cells). Centrifuge at 4°C, 900 rpm for 5 minutes in a low-temperature centrifuge and remove the supernatant.

[0081] (5) Mix the matrix gel and PBS in a 1:1 ratio, resuspend the mixed precipitate, and seed 50 μL per well on a 24-well cell plate, dropwise add the 3D semi-arched structure, and incubate in a cell culture incubator at 37°C for 30-60 minutes;

[0082] (6) When the matrix gel is completely solidified, the cells are divided into groups and 600 μL of the ENRP system or 600 μL of the ENRP system are added along the well wall of each group. The cells are then cultured at 37°C for 48 hours.

[0083] The size and growth status of intestinal organoids were observed under a microscope. Figure 2 As shown in A, the scale bar is 100μm. Compared with the ENR system treatment group, the intestinal organoid morphology of the ENRP system treatment group changed significantly. The volume of the intestinal organoids increased, and the "budding" organoids with the original crypt-villus structure were transformed into spherical organoids. At the same time, the ENRP system maintained the normal growth state of intestinal organoids in vitro. The morphology of the intestinal organoids in the ENR system treatment group and the ENRP system treatment group was statistically analyzed, and the results are shown as follows: Figure 2 As shown in B, the proportion of spherical organoids in the ENRP-treated group increased greatly, and the spherical organoids obtained were able to stably maintain their growth state in the ENRP-treated group and were able to undergo normal expansion and passaging, and their state was not affected after passaging.

[0084] Example 2: Validation of fetal-like intestinal stem cells in intestinal organoids

[0085] Real-time fluorescence quantitative PCR (qPCR) technology was used to verify the properties of intestinal organoids in the ENR system treatment group and the ENRP system treatment group.

[0086] 1. Expression of the fetal-like intestinal stem cell marker gene Sca1 (Stem Cell Antigen-1)

[0087] (1) Figure 3 As shown in A, compared with the ENR group, the expression level of fetal-like intestinal stem cell marker gene Sca1 (P<0.01) was significantly increased in the ENRP group.

[0088] (2) Transcriptome sequencing was performed on intestinal organoids in the ENR and ENRP groups. After the transcriptome data were statistically analyzed, GSEA enrichment analysis was performed. The results are as follows: Figure 3 As shown in B, the gene set related to fetal-like intestinal stem cells was significantly enriched in the intestinal organoids of the ENRP group.

[0089] (3) Perform differential expression analysis on the transcriptome data, and then perform cluster analysis on the differential gene sets. The results are as follows: Figure 3 As shown in C, the expression levels of fetal-like intestinal stem cell-related genes in the ENRP group were significantly increased compared with the ENR group.

[0090] The results of this part indicate that using the ENRP system to culture mouse intestinal organoids in vitro can increase the expression of fetal-like intestinal stem cell marker genes.

[0091] 2. Expression of marker genes of adult stem cells

[0092] (1) Figure 4 As shown in A, compared with the ENR group, the expression level of the adult stem cell marker gene Lgr5 in the ENRP group was significantly decreased (P<0.01).

[0093] (2) Transcriptome sequencing was performed on intestinal organoids in the ENR and ENRP groups. After the transcriptome data were statistically analyzed, GSEA enrichment analysis was performed. The results are as follows: Figure 4 As shown in B, the enrichment level of the adult stem cell-related gene Lgr5 set in the intestinal organoids of the ENRP group was significantly lower than that of the ENR group.

[0094] (3) Perform differential expression analysis on the transcriptome data, and then perform cluster analysis on the differential gene sets. The results are as follows: Figure 4 As shown in C, the expression level of adult stem cell-related gene Lgr5 in the ENRP group was significantly decreased compared with that in the ENR group.

[0095] (4) The expression levels of Hopx and Lrig1, marker genes of quiescent adult stem cells in intestinal organoids of the ENR and ENRP groups, were detected. Figure 5 As shown, compared with the ENR group, the expression levels of quiescent adult stem cell marker genes Hopx (P<0.01) and Lrig1 (P<0.01) in the ENRP group were also significantly reduced.

[0096] The above results show that after being cultured in the ENRP system, mouse intestinal organoids can significantly reduce the expression levels of active and quiescent adult stem cell marker genes. The spherical organoids obtained by in vitro culture in the ENRP system are in an undifferentiated state and can stably maintain their state.

[0097] 3. Expression of marker genes of Wnt signaling pathway

[0098] (1) Figure 6 As shown in A, compared with the ENR group, the expression level of Axin2, a marker gene of the Wnt signaling pathway, was significantly decreased in the ENRP group (P<0.01).

[0099] (2) Transcriptome sequencing was performed on intestinal organoids in the ENR and ENRP groups. After the transcriptome data were statistically analyzed, GSEA enrichment analysis was performed. The results are as follows: Figure 6 As shown in B, the enrichment of Wnt signaling pathway-related gene sets in intestinal organoids in the ENRP group was significantly lower than that in the ENR group.

[0100] (3) Perform differential expression analysis on the transcriptome data, and then perform cluster analysis on the differential gene sets. The results are as follows: Figure 6 As shown in C, the expression levels of genes related to the Wnt signaling pathway in the ENRP group were significantly decreased compared with those in the ENR group.

[0101] The Wnt signaling pathway is one of the key signaling pathways that maintains the self-renewal of intestinal adult stem cells and regulates their differentiation. The above results show that the Wnt signaling pathway is inhibited in the ENRP group, so the differentiation and proliferation of adult stem cells are also inhibited.

[0102] The above results fully prove that the "budding" organoids are in a differentiated state, while the spherical organoids are in an undifferentiated state, that is, intestinal organoids rich in fetal-like intestinal stem cells are obtained and cultured by the method provided by the present invention.

[0103] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. Use of prostaglandin E2 in maintaining the fetal-like intestinal stem cell state, characterized in that: The application uses a culture medium to convert intestinal epithelial cells into fetal-like intestinal stem cells in vitro and maintain the fetal-like intestinal stem cell state; The culture medium comprises a basal culture medium, a serum replacement, antibiotics, glutamate / ester, a HEPES buffer, epidermal growth factor EGF, Noggin, R-spondin and prostaglandin E2; The concentration of the prostaglandin E2 in the culture medium is 3-7 μg / mL.

2. The use according to claim 1, characterized in that: The intestinal epithelial cells originate from the small intestine.

3. The use according to claim 1, characterized in that: The intestinal epithelial cells are mouse intestinal epithelial cells.

4. The use according to claim 1, characterized in that: The basic culture medium is DMEM / F12 complete culture medium.

5. The use according to claim 1, characterized in that: The serum replacements were N2 and B27.

6. The use according to claim 1, characterized in that: The antibiotics are penicillin and streptomycin.

7. The use according to claim 1, characterized in that: The glutamate / ester is Glutamax, and the concentration of the Glutamax is 2 mM.

8. The use according to claim 1, characterized in that: The concentration of the HEPES buffer is 1M.

9. The use according to claim 1, characterized in that: The concentration of the epidermal growth factor EGF is 30-70 ng / mL, the concentration of the Noggin is 50-150 ng / mL, and the concentration of the R-spondin is 500-1000 ng / mL.

10. A culture medium for maintaining a fetal-like intestinal stem cell state, comprising a basal culture medium, a serum replacement, antibiotics, glutamate / ester, a HEPES buffer, epidermal growth factor EGF, Noggin, R-spondin and prostaglandin E2, characterized in that: The basal culture medium is DMEM / F12 complete culture medium; The serum replacements are N2 and B27; The glutamate / ester is Glutamax, and the concentration of Glutamax is 2mM; The concentration of the HEPES buffer is 1M; The concentration of the epidermal growth factor EGF is 30-70 ng / mL, the concentration of the Noggin is 50-150 ng / mL, and the concentration of the R-spondin is 500-1000 ng / mL; The concentration of prostaglandin E2 is 3-7 μg / mL.

11. A method for obtaining fetal-like intestinal stem cells in vitro, characterized in that: The intestinal crypts encapsulated in the matrix gel are placed in the culture medium of claim 10 for culture.

12. The method according to claim 11, characterized in that: The intestinal crypts were isolated from the small intestine.

13. The method according to claim 11, characterized in that: The culture was carried out under sterile conditions, 5% CO2 and 37°C.

14. A method for culturing fetal-like intestinal stem cells, characterized in that: The fetal-like intestinal stem cells encapsulated in the matrix gel are placed in the culture medium of claim 10 for culture, wherein the fetal-like intestinal stem cells are obtained by the method of any one of claims 11-13.

15. The culture method according to claim 14, characterized in that: The culture was carried out under sterile conditions, 5% CO2 and 37°C.

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