Differentiation inhibitor for stem cell culture, method for producing differentiation inhibitor for stem cell culture, method for inhibiting differentiation of stem cells, cell culture medium for stem cell culture, method for producing cell culture medium for stem cell culture, and method for culturing stem cells maintained in undifferentiated state

Abstract

To provide a differentiation inhibitor for stem cell culture which can inhibit differentiation of stem cells and increase cell yield when the stem cells are subjected to stirred culture.SOLUTION: A differentiation inhibitor for stem cell culture comprising, as an active ingredient, an alkaline extract protein of tea leaves. It is preferable that the tea leaves are residues obtained after hot-water extraction treatment of tea leaves. It is also preferable that the alkaline extract protein has a molecular weight of 3000 or more. Furthermore, it is preferable that the tea leaves are green tea.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a technique for culturing stem cells, and in particular to a differentiation inhibitor for stem cell culture. [Background technology]

[0002] Traditionally, induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells) have faced the problem of being difficult to grow while maintaining good quality. In other words, since these stem cells are generally mass-cultured in an undifferentiated state and then induced to differentiate into the desired cells for use, it is desirable that they remain undifferentiated during the proliferation stage.

[0003] However, there are many unknowns about the mechanism that maintains stem cells in an undifferentiated state, and it has not been easy to mass-cultivate high-quality undifferentiated stem cells or to maintain the resulting high-quality stem cells in an undifferentiated state. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2014-50356 [Patent Document 2] Japanese Patent Publication No. 2020-5517 Summary of the Invention [Problem to be solved by the invention]

[0005] In addition, spinner culture may be performed using a reactor (e.g., 30 mL) to culture stem cells in large quantities. However, spinner culture has the problem of being less efficient at growing cells than adherent culture. Specifically, for example, by culturing iPS cells in an adherent culture for four days, the cell number could be increased by more than 60 times, but when the same iPS cells were cultured in a stirred culture, the cell proliferation rate was only a few times higher.

[0006] On the other hand, when culturing stem cells on a large scale using adherent culture, it is necessary to secure a large culture area, which makes industrial application difficult. For this reason, there has been a demand for improving the yield of spinner culture in order to cultivate stem cells industrially on a large scale.

[0007] Therefore, the inventors conducted extensive research and succeeded in developing a differentiation inhibitor for stem cell culture that can inhibit stem cell differentiation and increase the cell yield when stem cells are cultured in agitation, by using protein components obtained by alkaline extraction of tea leaves, thereby completing the present invention.

[0008] Patent Document 1 describes an agent for inducing differentiation of stem cells into ectodermal cells using black tea extract. However, this document does not mention the use of protein components obtained by alkaline extraction of tea leaves, nor does it mention the inhibition of stem cell differentiation. Patent Document 2 describes a cell culture medium using a black tea infusion extract. However, this document also does not mention the use of protein components obtained by alkaline extraction of tea leaves. Although this document states that black tea components have been confirmed to promote cell proliferation and inhibit differentiation, it does not state the basis for this confirmation.

[0009] The present invention has been made in consideration of the above circumstances, and aims to provide a differentiation inhibitor for stem cell culture that can inhibit stem cell differentiation and increase the cell yield when stem cells are cultured in agitation, a method for producing a differentiation inhibitor for stem cell culture, a method for inhibiting stem cell differentiation, a cell culture medium for stem cell culture, a method for producing a cell culture medium for stem cell culture, and a method for culturing stem cells to maintain their undifferentiated state. [Means for solving the problem]

[0010] In order to achieve the above object, the differentiation inhibitor for stem cell culture of the present invention is configured to contain alkaline-extracted protein from tea leaves as an active ingredient. Furthermore, it is preferable that the differentiation inhibitor for stem cell culture of the present invention is configured such that the tea leaves are residues remaining after tea leaves have been subjected to a hot water extraction treatment.

[0011] Furthermore, it is preferable that the differentiation inhibitor for stem cell culture of the present invention is configured so that the molecular weight of the alkali-extracted protein is 3,000 or more. Furthermore, it is preferable that the differentiation inhibitor for stem cell culture of the present invention is configured such that the tea leaves are green tea. Furthermore, it is also preferable that the differentiation inhibitor for stem cell culture of the present invention is configured by combining various of the above-mentioned differentiation inhibitors for stem cell culture.

[0012] The method for producing the differentiation inhibitor for stem cell culture of the present invention is a method in which tea leaves are extracted with hot water, the residue is extracted with alkali, and the resulting protein is contained as an active ingredient. Furthermore, it is preferable that the method for producing the differentiation inhibitor for stem cell culture of the present invention is a method in which the residue is subjected to alkaline extraction, the resulting extract is then ultrafiltered, and the protein contained therein is one with a molecular weight of 3000 or more.

[0013] The method of inhibiting stem cell differentiation of the present invention involves subjecting tea leaves to a hot water extraction treatment, followed by an alkaline extraction treatment of the residue, and adding a differentiation inhibitor containing the resulting protein as an active ingredient to a cell culture medium, and culturing stem cells in the cell culture medium. Furthermore, it is preferable that the method of inhibiting stem cell differentiation of the present invention is a method in which the residue is subjected to alkaline extraction, the resulting extract is ultrafiltered, and the protein having a molecular weight of 3000 or more is contained in the differentiation inhibitor.

[0014] The cell culture medium for stem cell culture of the present invention is configured to contain alkaline-extracted protein from tea leaves as an active ingredient. It is also preferable that the cell culture medium for stem cell culture of the present invention is configured so that the alkali-extracted protein has a molecular weight of 3,000 or more. Furthermore, it is also preferable that the cell culture medium for stem cell culture of the present invention contains the alkali-extracted protein at a concentration of 1 to 400 μg / mL.

[0015] The method for producing a cell culture medium for stem cell culture of the present invention is a method in which tea leaves are subjected to hot water extraction, the residue is subjected to alkaline extraction, and the resulting protein is contained as an active ingredient. Furthermore, it is also preferred that the method for producing a cell culture medium for stem cell culture of the present invention comprises subjecting the residue to alkaline extraction treatment, and then ultrafiltrating the resulting extract to contain proteins with a molecular weight of 3,000 or more.

[0016] The method of the present invention for culturing stem cells in an undifferentiated state involves subjecting the residue of tea leaves to an alkaline extraction treatment after hot water extraction, adding a differentiation inhibitor containing the resulting protein as an active ingredient to a cell culture medium, and culturing stem cells in the cell culture medium. [Effects of the Invention]

[0017] According to the present invention, it is possible to provide a differentiation inhibitor for stem cell culture that can inhibit stem cell differentiation and increase the cell yield when stem cells are cultured in agitation, a method for producing a differentiation inhibitor for stem cell culture, a method for inhibiting stem cell differentiation, a cell culture medium for stem cell culture, a method for producing a cell culture medium for stem cell culture, and a method for culturing stem cells to maintain their undifferentiated state. [Brief explanation of the drawings]

[0018] [Figure 1] FIG. 1 shows the results of Test 1 (Evaluation Method A) for confirming the differentiation inhibitory effect of a differentiation inhibitor for stem cell culture, etc., according to an embodiment of the present invention. [Figure 2] FIG. 1 shows the results of Test 1 (Evaluation Method B) for confirming the differentiation inhibitory effect of a differentiation inhibitor for stem cell culture, etc., according to an embodiment of the present invention. [Figure 3]FIG. 1 shows the results of Test 2 (culture period: 3 days) to confirm the effect of increasing yield by agitated cell culture using a differentiation inhibitor for stem cell culture according to an embodiment of the present invention. [Figure 4] FIG. 1 shows the results of Test 2 (culture period: 5 days) to confirm the effect of increasing yield by agitated cell culture using a differentiation inhibitor for stem cell culture according to an embodiment of the present invention. [Figure 5] FIG. 1 shows the results of Test 3 (evaluation method A, without ultrafiltration) for confirming the differentiation inhibitory effect of a differentiation inhibitor for stem cell culture, etc., according to an embodiment of the present invention. [Figure 6] FIG. 1 shows the results of Test 3 (evaluation method B, without ultrafiltration) for confirming the differentiation inhibitory effect of a differentiation inhibitor for stem cell culture, etc., according to an embodiment of the present invention. [Figure 7] FIG. 1 shows the results of Test 4 (cultivation period: 5 days, no ultrafiltration) to confirm the effect of increasing yield by agitation culture of cells using a differentiation inhibitor for stem cell culture according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0019] Hereinafter, embodiments of the differentiation inhibitor for stem cell culture, the method for producing the differentiation inhibitor for stem cell culture, the method for inhibiting stem cell differentiation, the cell culture medium for stem cell culture, the method for producing the cell culture medium for stem cell culture, and the method for maintaining undifferentiated stem cells in culture according to the present invention will be described in detail. However, the present invention is not limited to the specific details of the following embodiments and the examples described below.

[0020] The differentiation inhibitor for stem cell culture of this embodiment can be added to the culture medium during stem cell culture to obtain the effect of inhibiting stem cell differentiation and the effect of increasing the yield by agitating culture of stem cells. The differentiation inhibitor for stem cell culture of this embodiment is characterized by containing alkaline-extracted protein from tea leaves as an active ingredient.

[0021] The tea leaves are preferably residues remaining after tea leaves have been subjected to hot water extraction treatment. It is also preferable to use tea leaves that have been extracted with hot water and then dried. The hot water extraction treatment can be selected as appropriate, but for example, treatment can be carried out in heated water (pure water) at 70°C to 90°C for 2 minutes to 180 minutes.

[0022] The residue left after hot water extraction of tea leaves is generally treated as waste, although some of it is used as fertilizer or animal feed. In this situation, the differentiation inhibitor for stem cell culture of this embodiment makes it possible to obtain the effect of inhibiting stem cell differentiation and the effect of increasing yield through agitated culture of stem cells by using the residue left after hot water extraction of tea leaves. It is also possible to use tea leaves that have not been subjected to hot water extraction treatment or that have been dried without being subjected to hot water extraction treatment.

[0023] The alkali-extracted protein from tea leaves means a protein obtained by soaking tea leaves in an alkali solution and dissolving the tea leaves in the alkali solution. Specifically, for example, it can be obtained as follows. The residue remaining after the hot water extraction treatment of tea leaves is mixed with an aqueous sodium hydroxide solution and stirred for a predetermined period of time using a stirrer. The resulting composition is then centrifuged and the supernatant is collected to obtain a tea leaf extract containing alkaline-extracted proteins of tea leaves.

[0024] The molecular weight of the alkali-extracted protein in the differentiation inhibitor for stem cell culture of this embodiment is preferably 3,000 or more. The differentiation inhibitor for stem cell culture of this embodiment, which is made by ultrafiltering tea leaf extract to contain only components with a molecular weight of 3000 or more, can further improve the yield-increasing effect of agitated culture of stem cells compared to those containing components with a molecular weight of less than 3000.

[0025] Furthermore, tea leaves contain catechins such as epigallocatechin, which are known to be cytotoxic. Therefore, differentiation inhibitors for stem cell culture, which are made by ultrafiltrating tea leaf extract to contain only components with a molecular weight of 3000 or more, can be more suitably used in cell culture because low molecular weight components such as catechins have been removed.

[0026] Furthermore, the differentiation inhibitor for stem cell culture of this embodiment preferably contains alkali-extracted protein at a concentration of 0.1 to 200 mg / mL. This is because the differentiation inhibitor for stem cell culture of this embodiment, which contains alkali-extracted protein at a concentration of 0.1 to 200 mg / mL, is expected to have an excellent effect of inhibiting stem cell differentiation and an effect of increasing the yield by agitating culture of stem cells.

[0027] Here, the cell culture medium for stem cell culture of this embodiment, which will be described later, is prepared by adding the differentiation inhibitor for stem cell culture of this embodiment to a separately prepared culture medium, and adjusting the final concentration of the alkaline-extracted protein to fall within a predetermined concentration range. In this case, if the concentration of the differentiation inhibitor for stem cell culture is less than 0.1 mg / mL, a large amount must be mixed with the separately prepared culture medium, which may result in excessive dilution of the components originally contained in the culture medium and a decrease in culture performance.Furthermore, if the concentration exceeds 200 mg / mL, the concentration is excessively high for a protein solution, which may result in changes in quality, such as precipitation of components. The cell culture medium for stem cell culture of this embodiment, prepared by mixing the differentiation inhibitor for stem cell culture of this embodiment with a separately prepared culture medium, is expected to have an excellent effect of inhibiting stem cell differentiation and an effect of increasing yield through agitated culture of stem cells.

[0028] Furthermore, from this perspective, the differentiation inhibitor for stem cell culture of this embodiment preferably contains alkali-extracted protein at a concentration of 0.3 to 175 mg / mL, more preferably at a concentration of 0.5 to 150 mg / mL, even more preferably at a concentration of 1 to 100 mg / mL, and particularly preferably at a concentration of 1.5 to 50 mg / mL.

[0029] The type of tea leaves in the differentiation inhibitor for stem cell culture of this embodiment is not particularly limited, but green tea is preferred. The differentiation inhibitor for stem cell culture of this embodiment, which is made from green tea as a raw material, can exhibit an excellent effect of inhibiting stem cell differentiation and an effect of increasing the yield of stem cells through agitated culture.

[0030] As stem cells to be cultured using the differentiation inhibitor for stem cell culture of this embodiment, induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), and the like can be suitably used.

[0031] The method for producing a differentiation inhibitor for stem cell culture of this embodiment is characterized by subjecting the residue after hot water extraction of tea leaves to alkaline extraction, and incorporating the resulting protein as an active ingredient. Furthermore, in the method for producing the differentiation inhibitor for stem cell culture of this embodiment, it is preferable that after the residue is subjected to alkaline extraction treatment, the resulting extract is ultrafiltered to contain proteins with a molecular weight of 3000 or more. According to the manufacturing method of the differentiation inhibitor for stem cell culture of this embodiment, it is possible to preferably obtain a differentiation inhibitor for stem cell culture that has excellent effects of inhibiting stem cell differentiation and increasing the yield by agitating culture of stem cells.

[0032] The method for inhibiting stem cell differentiation in this embodiment is characterized by subjecting the residue after hot water extraction of tea leaves to alkaline extraction, adding a differentiation inhibitor containing the resulting protein as an active ingredient to a cell culture medium, and culturing stem cells in the cell culture medium. Furthermore, in the method for inhibiting differentiation of stem cells of this embodiment, it is preferable that after alkaline extraction of the residue, the resulting extract is ultrafiltered to contain proteins with a molecular weight of 3000 or more in the differentiation inhibitor.

[0033] Furthermore, in the method for inhibiting stem cell differentiation of this embodiment, the concentration of the protein contained in the cell culture medium is preferably 1 to 400 μg / mL, more preferably 3 to 200 μg / mL, even more preferably 5 to 150 μg / mL, and particularly preferably 10 to 100 μg / mL. According to the method for inhibiting stem cell differentiation of this embodiment, it is possible to preferably inhibit the differentiation of stem cells when culturing the stem cells.

[0034] The cell culture medium for stem cell culture of this embodiment is characterized by containing alkaline-extracted protein from tea leaves as an active ingredient. Furthermore, in the cell culture medium for stem cell culture of this embodiment, the molecular weight of the alkali-extracted protein is preferably 3,000 or more.

[0035] Furthermore, the cell culture medium for stem cell culture of this embodiment preferably contains the alkali-extracted protein at a concentration of 1 to 400 μg / mL, more preferably 3 to 200 μg / mL, even more preferably 5 to 150 μg / mL, and particularly preferably 10 to 100 μg / mL. According to the cell culture medium for stem cell culture of this embodiment, it is possible to obtain an effect of inhibiting differentiation of stem cells when culturing stem cells.

[0036] The method for producing a cell culture medium for stem cell culture of this embodiment is characterized by subjecting the residue after hot water extraction of tea leaves to alkaline extraction, and incorporating the obtained protein as an active ingredient. Furthermore, in the method for producing a cell culture medium for stem cell culture of this embodiment, it is preferable that after the residue is subjected to an alkaline extraction treatment, the resulting extract is ultrafiltered to contain proteins with a molecular weight of 3000 or more.

[0037] Furthermore, in the method for producing a cell culture medium for stem cell culture of this embodiment, the concentration of the protein contained in the cell culture medium for stem cell culture is preferably 1 to 400 μg / mL, more preferably 3 to 200 μg / mL, even more preferably 5 to 150 μg / mL, and particularly preferably 10 to 100 μg / mL. According to the method for producing a cell culture medium for stem cell culture of this embodiment, it is possible to preferably obtain a cell culture medium for stem cell culture that has the effect of inhibiting stem cell differentiation when culturing stem cells.

[0038] The method for culturing stem cells in an undifferentiated state in this embodiment is characterized by subjecting the residue after hot water extraction of tea leaves to alkaline extraction, adding a differentiation inhibitor containing the resulting protein as an active ingredient to a cell culture medium, and culturing stem cells in the cell culture medium. According to the method for culturing stem cells to maintain their undifferentiated state in this embodiment, stem cells can be cultured in an appropriate manner while maintaining the undifferentiated state of the stem cells. [Example]

[0039] [Test 1] A test to confirm the differentiation inhibitory effect of the differentiation inhibitor for stem cell culture according to an embodiment of the present invention was carried out as follows.

[0040] <Extraction of tea leaf-derived protein> The tea leaves were extracted with hot water, and the residue was then subjected to alkaline extraction to obtain a protein-containing tea leaf extract sample. Specifically, Sencha tea was used as the tea leaves, and the residue after hot water extraction was dried. Next, 5 g of this residue was mixed with 70 mL of a 0.05 M aqueous solution of NaOH (Fujifilm Wako Pure Chemical Industries, Ltd.), and the mixture was stirred at 60° C. for 3 hours using a hot stirrer (As One Corporation). Furthermore, the obtained composition was centrifuged (6000×g, 60 minutes) and the supernatant was collected to obtain Sample 1 of tea leaf extract containing tea leaf-derived proteins.

[0041] <Measurement of protein concentration in tea leaf extract> The protein concentration in sample 1 of the tea leaf extract was measured by the BCA method. Specifically, a protein concentration measurement kit (TaKaRa BCA Protein Assay Kit, Takara Bio Inc.) was used. Following the manufacturer's instructions, equal volumes of the prepared BCA reaction solution and the sample to be measured, appropriately diluted with phosphate buffer (Nacalai Tesque, Inc.), were mixed in a 96-well plate (351172, Corning Incorporated) and allowed to stand at 37°C for 2 hours. The absorbance was then measured using a microplate reader (Corona Electric Co., Ltd.) at a dominant wavelength of 562 nm and a sub-wavelength of 447 nm to determine the protein concentration. The bovine serum albumin (BSA) included in the kit was used for the calibration curve. As a result, the concentration of tea leaf-derived protein contained in tea leaf extract sample 1 was 20.4 mg / mL.

[0042] <Preparation of components with molecular weight cutoff of 3000 or more by ultrafiltration> Next, tea leaf extract sample 1 was ultrafiltered to contain tea leaf-derived proteins with a molecular weight of 3000 or more. Specifically, 500 μL / container of tea leaf extract sample 1 was placed in an ultrafiltration filter (Nanosep®, molecular weight cutoff 3000, Cytiva) and centrifuged (11000×g, 45 minutes) to remove low molecular weight components.

[0043] At this time, an equal volume of 0.05M NaOH aqueous solution to the volume of the liquid transferred to the low molecular weight fraction was added to the high molecular weight component side, and centrifugation was repeated 15 times. 150μL of the resulting high molecular weight component liquid was then mixed with 350μL of 0.05M NaOH aqueous solution to obtain Sample 2, a tea leaf extract from which the low molecular weight components had been removed. The calculated residual rate of low molecular weight components was 0.0043% before ultrafiltration. The concentration of tea leaf-derived protein in the high molecular weight components was measured by the BCA method and found to be 5.02 mg / mL.

[0044] <Cell adhesion culture and evaluation of differentiation inhibitory effects> Next, cells were cultured in adherent culture using tea leaf extract sample 2, and the differentiation-inhibitory effect of tea leaf extract sample 2 was evaluated. Specifically, iPS cells (1231A3 strain, Kyoto University) were used.

[0045] The cells were suspended in a medium (StemFit, Ajinomoto Co., Inc.) containing 10 mM Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.) and 15.7 mg / mL iMatrix511 (Nippi Corporation), and plated at 1.5 × 10 cells per well onto five 48-well plates (cell adhesion-treated, Corning). 3 The cells were seeded at 300 μL / well, and the day after seeding, the medium was replaced with one that did not contain Y-27632 or iMatrix-511. Then, cell culture was performed by adding tea leaf extract sample 2 to the medium so that the final concentrations of tea leaf-derived protein were 5.9 μg / mL, 29.5 μg / mL, 145.1 μg / mL, and 672.7 μg / mL, respectively. Alternatively, cell culture was performed using a medium without adding tea leaf extract sample 2, in which the final concentration of tea leaf-derived protein was 0 μg / mL.

[0046] The timing of addition of tea leaf extract sample 2 was 5, 9, and 16 days after the start of culture, and the cells were cultured for a total of 12 days, from 5 to 7 days, from 9 to 14 days, and from 16 to 21 days, respectively. At the end of the 7th and 14th days after the start of culture, the cells were detached from the 48-well plate using a detachment solution (TrypLE Select CTS, Thermo Fisher Scientific) diluted 2-fold with phosphate buffer containing 0.5 mM EDTA (Nacalai Tesque, Inc.), and the cells were again transferred to the 48-well plate at a density of 1.5 × 10 3 The cells were replated at 300 μL / well. Then, at the end of the culture on the 21st day after the start of the culture, the cells were detached from the 48-well plate using the same detachment solution.

[0047] In this way, tea leaf-derived protein was added to the medium so that the final concentrations were 0 μg / mL, 5.9 μg / mL, 29.5 μg / mL, and 145.1 μg / mL, respectively, and cell culture was performed, and cells were obtained after 12 days of culture. On the other hand, when tea leaf-derived protein was added to the medium at a concentration of 672.7 μg / mL and cells were cultured, the cells died on the seventh day, and it was not possible to obtain cells after 12 days of culture.

[0048] The inhibitory effect of tea leaf extract sample 2 on differentiation of cultured cells was then evaluated using undifferentiation markers with flow cytometry. In this case, evaluation was performed using two methods: evaluation method A (using the SSEA-1 positive rate) and evaluation method B (using the double positive rate of SSEA-4 and TRA1-60R). In evaluation method A, a lower positive rate indicates higher quality, while in evaluation method B, a higher double positive rate indicates higher quality.

[0049] Specifically, cells were stained with the antibody for evaluation method A (Alexa Flour 488®-labeled anti-SSEA-1 antibody, R&D Systems) and the antibodies for evaluation method B (APC-labeled anti-SSEA-4 antibody, PE-labeled anti-TRA1-60R antibody, Biolegend), and the fluorescence intensity was measured using a flow cytometer (CytoFLEX S, Beckman Coulter). Cells that were not antibody-stained were used as the standard for determining whether the cells were positive or negative. The results are shown in Figures 1 and 2.

[0050] As shown in Figure 1, in Comparative Example 1, where the concentration of tea leaf-derived protein was 0 μg / mL, the SSEA-1 positive rate was 6.99%, in Example 1, where the concentration of tea leaf-derived protein was 5.9 μg / mL, the SSEA-1 positive rate was 6.27%, in Example 2, where the concentration of tea leaf-derived protein was 29.5 μg / mL, the SSEA-1 positive rate was 5.58%, and in Example 3, where the concentration of tea leaf-derived protein was 145.1 μg / mL, the SSEA-1 positive rate was 6.3%.

[0051] Furthermore, as shown in Figure 2, in Comparative Example 1, where the concentration of tea leaf-derived protein was 0 μg / mL, the double positive rate of SSEA-4 and TRA1-60R was 82.22%, in Example 1, where the concentration of tea leaf-derived protein was 5.9 μg / mL, the double positive rate of SSEA-4 and TRA1-60R was 93.76%, in Example 2, where the concentration of tea leaf-derived protein was 29.5 μg / mL, the double positive rate of SSEA-4 and TRA1-60R was 95.4%, and in Example 3, where the concentration of tea leaf-derived protein was 145.1 μg / mL, the double positive rate of SSEA-4 and TRA1-60R was 95.76%.

[0052] That is, in Examples 1 to 3, in which tea leaf extract sample 2 was added so that the tea leaf-derived protein concentration was in the range of 5.9 μg / mL to 145.1 μg / mL, the positive rate in Evaluation Method A decreased, while the double positive rate in Evaluation Method B increased, compared to Comparative Example 1, in which tea leaf extract sample 2 was not added. These results demonstrate that tea leaf extract sample 2 can suppress the phenomenon of uncontrolled differentiation of iPS cells and improve the quality of iPS cell culture.

[0053] [Test 2] A test was conducted as follows to confirm the effect of increasing yield by agitation culture of cells using a differentiation inhibitor for stem cell culture according to an embodiment of the present invention.

[0054] <Agitated cell culture using tea leaf extract-containing medium> iPS cells were cultured in a medium containing Sample 2 of the tea leaf extract from Test 1. Specifically, iPS cells (1231A3 strain, Kyoto University) were suspended in a medium (StemFit, Ajinomoto Co., Inc.) containing 10 mM Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.) and seeded into four 30 mL culture reactors (Able Co., Ltd.). 6 In Comparative Example 3 and Example 5, 1.5 × 10 6 The seeding rate was 30 mL / reactor.

[0055] The day after seeding, the medium was replaced with one not containing Y-27632. For Examples 4 and 5, tea leaf extract Sample 2 was added to the medium so that the final tea leaf-derived protein concentration was 29.5 μg / mL, and cell culture was performed. For Comparative Examples 2 and 4, cell culture was performed without adding tea leaf extract Sample 1 to the medium.

[0056] After further culturing for 2 days (Comparative Example 2 and Example 4) or 4 days (Comparative Example 3 and Example 5), the aggregated cells were dissociated into single cells using a detachment solution (TrypLE Select CTS) diluted 2-fold with phosphate buffer containing 0.5 mM EDTA, and the viable cell count was measured using a cell counter (NucleoCounter® NC-200™, Chemometec). The results are shown in Figures 3 and 4.

[0057] As shown in FIG. 3, in Comparative Example 2 where the tea leaf-derived protein concentration was 0 μg / mL after 3 days of culture, the viable cell count was 1.263 × 10 7 The multiplication rate was 3.27 times. In contrast, in Example 4, where the tea leaf-derived protein concentration was 29.5 μg / mL, the number of viable cells was 1.626 × 10 7 The multiplication rate was 4.21 times.

[0058] Furthermore, as shown in FIG. 4, in Comparative Example 3 where the tea leaf-derived protein concentration was 0 μg / mL after 5 days of culture, the viable cell count was 1.302 × 10 7 The multiplication rate was 8.68 times. In contrast, in Example 5, where the tea leaf-derived protein concentration was 29.5 μg / mL, the number of viable cells was 1.998 × 10 7 The multiplication rate was 13.31 times.

[0059] In other words, when comparing experiments with the same number of culture days, those in which tea leaf-derived protein was added to a concentration of 29.5 μg / mL (Examples 4 and 5) showed a higher proliferation rate than those in which no tea leaf-derived protein was added (Comparative Examples 3 and 4). These results demonstrate that tea leaf extract sample 2 can increase the yield of cells by culturing them under agitation. Furthermore, this result means that a smaller amount of medium is required to obtain the desired number of cells, confirming that the differentiation inhibitor for stem cell culture of this embodiment is industrially useful.

[0060] [Test 3] A test (without ultrafiltration) to confirm the differentiation inhibitory effect of the differentiation inhibitor for stem cell culture according to an embodiment of the present invention was carried out as follows.

[0061] <Cell adhesion culture using medium containing tea leaf extract (without ultrafiltration)> Using sample 1 of the tea leaf extract from Test 1 (without ultrafiltration), cells were cultured in adherent culture, and the differentiation-inhibitory effect of sample 1 of the tea leaf extract was evaluated. Specifically, iPS cells (1231A3 strain, Kyoto University) were used.

[0062] The cells were suspended in a medium (StemFit, Ajinomoto Co., Inc.) containing 10 mM Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.) and 15.7 mg / mL iMatrix511 (Nippi Corporation), and plated at 1.5 × 10 cells per well onto two 48-well plates (cell adhesion-treated, Corning). 3The cells were seeded at 300 μL / well, and the day after seeding, the medium was replaced with one that did not contain Y-27632 or iMatrix-511. Then, cell culture was performed by adding tea leaf extract sample 1 to the medium so that the final concentration of tea leaf-derived protein was 29.5 μg / mL.Also, cell culture was performed using a medium without adding tea leaf extract sample 1, in which the final concentration of tea leaf-derived protein was 0 μg / mL.

[0063] The timing of addition of tea leaf extract sample 1 was on the first day (the day culture was started) and on the ninth day after the start of culture, and the cells were cultured for a total of 11 days, from the first to the seventh day and from the ninth to the fourteenth day, respectively. At the end of the 7th and 14th days after the start of culture, the cells were detached from the 48-well plate using a detachment solution (TrypLE Select CTS, Thermo Fisher Scientific) diluted 2-fold with phosphate buffer containing 0.5 mM EDTA (Nacalai Tesque, Inc.), and the cells were again transferred to the 48-well plate at a density of 1.5 × 10 3 The cells were replated at 300 μL / well.

[0064] Then, at the end of the culture on the 14th day after the start of the culture, the cells were detached from the 48-well plate using the same detachment solution. In this manner, tea leaf-derived protein was added to the medium so that the final concentrations thereof became 0 μg / mL and 29.5 μg / mL, respectively, and cell culture was carried out, and cells were obtained after 11 days of culture.

[0065] Then, the inhibitory effect of tea leaf extract Sample 1 on differentiation of cultured cells was evaluated using undifferentiation markers using flow cytometry, as in Test 1. Specifically, cells were stained with the antibody for evaluation method A (Alexa Flour 488®-labeled anti-SSEA-1 antibody, R&D Systems) and the antibodies for evaluation method B (APC-labeled anti-SSEA-4 antibody, PE-labeled anti-TRA1-60R antibody, Biolegend), and the fluorescence intensity was measured using a flow cytometer (CytoFLEX S, Beckman Coulter). Cells that were not antibody-stained were used as the standard for determining whether the cells were positive or negative. The results are shown in Figures 5 and 6.

[0066] As shown in Figure 5, in Comparative Example 4, where the concentration of tea leaf-derived protein was 0 μg / mL, the SSEA-1 positive rate was 5.54%, and in Example 6, where the concentration of tea leaf-derived protein was 29.5 μg / mL, the SSEA-1 positive rate was 3.56%. Furthermore, as shown in Figure 6, in Comparative Example 4, where the concentration of tea leaf-derived protein was 0 μg / mL, the double positive rate of SSEA-4 and TRA1-60R was 77.55%, and in Example 6, where the concentration of tea leaf-derived protein was 29.5 μg / mL, the double positive rate of SSEA-4 and TRA1-60R was 89.44%.

[0067] That is, in Example 6, in which tea leaf extract sample 1 was added so that the tea leaf-derived protein concentration was 29.5 μg / mL, the positive rate in Evaluation Method A decreased, while the double positive rate in Evaluation Method B increased, compared to Comparative Example 4, in which tea leaf extract sample 1 was not added. These results demonstrate that tea leaf extract Sample 1 can suppress the phenomenon of uncontrolled differentiation of iPS cells and improve the quality of iPS cell culture.

[0068] [Test 4] A test (without ultrafiltration) to confirm the effect of the differentiation inhibitor for stem cell culture according to an embodiment of the present invention on increasing yield by agitation culture was carried out as follows.

[0069] <Agitated cell culture using a medium containing tea leaf extract (without ultrafiltration)> iPS cells were cultured in a spinner culture medium containing Sample 1 of the tea leaf extract from Test 1 (without ultrafiltration). Specifically, iPS cells (1231A3 strain, Kyoto University) were suspended in a medium (StemFit, Ajinomoto Co., Inc.) containing 10 mM Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.), and placed in two 30 mL culture reactors (Able Co., Ltd.) at a concentration of 1.5 × 10 6 The seeding rate was 30 mL / reactor.

[0070] The day after seeding, 70% of the medium was replaced with a medium not containing Y-27632, and cell culture was performed by adding tea leaf extract sample 1 to the medium so that the final concentration of tea leaf-derived protein was 29.5 μg / mL for Example 7. For Comparative Example 5, cell culture was performed without adding tea leaf extract sample 1 to the medium.

[0071] After further culturing for 4 days, the aggregated cells were dissociated into single cells using a detachment solution (TrypLE Select CTS) diluted 2-fold with phosphate buffer containing 0.5 mM EDTA, and the viable cell count was measured using a cell counter (NucleoCounter® NC-200™, Chemometec). The results are shown in Figure 7.

[0072] As shown in FIG. 7, in Comparative Example 5 where the tea leaf-derived protein concentration was 0 μg / mL after 5 days of culture, the viable cell count was 1.215 × 10 7 The multiplication rate was 8.09 times. In contrast, in Example 7, where the tea leaf-derived protein concentration was 29.5 μg / mL, the number of viable cells was 1.686 × 10 7 The multiplication rate was 11.23 times.

[0073] That is, in Example 7, in which the tea leaf-derived protein was added so that the concentration was 29.5 μg / mL, the cell count increased significantly compared to Comparative Example 5, in which no tea leaf-derived protein was added. These results demonstrate that even when using tea leaf extract that does not undergo ultrafiltration and contains components with a molecular weight of 3000 or less, the yield can be increased by culturing cells under agitation, and the amount of culture medium used can be reduced.

[0074] Furthermore, in Example 5 of Test 2, where the culture period was 5 days, the proliferation rate was improved by 53% compared to Comparative Example 3, while in Example 7 of Test 4, where the culture period was also 5 days, the proliferation rate was improved by 39% compared to Comparative Example 5. This indicates that the differentiation inhibitor for stem cell culture obtained by ultrafiltration has a superior effect on increasing cell yield through agitated cell culture compared to the differentiation inhibitor for stem cell culture obtained without ultrafiltration.

[0075] The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope of the present invention. For example, it is possible to use tea leaves that have not been subjected to hot water extraction treatment. [Industrial Applicability]

[0076] The present invention can be suitably used to effectively utilize the residue after hot water extraction of tea leaves, much of which is considered waste, to produce a differentiation inhibitor for stem cell culture that has excellent effects in inhibiting stem cell differentiation and increasing the yield through agitated cultivation of stem cells.

Claims

1. A differentiation inhibitor for stem cell culture, characterized by containing alkaline-extracted protein from tea leaves as an active ingredient.

2. 2. The differentiation inhibitor for stem cell culture according to claim 1, wherein the tea leaves are a residue obtained after hot water extraction of tea leaves.

3. 3. The differentiation inhibitor for stem cell culture according to claim 1, wherein the molecular weight of the alkali-extracted protein is 3,000 or more.

4. 3. The differentiation inhibitor for stem cell culture according to claim 1 or 2, wherein the tea leaves are green tea.

5. A method for producing a differentiation inhibitor for stem cell culture, characterized by subjecting tea leaves to hot water extraction, followed by alkali extraction of the residue, and incorporating the resulting protein as an active ingredient.

6. The method for producing a differentiation inhibitor for stem cell culture described in claim 5, characterized in that after alkaline extraction treatment of the residue, the obtained extract is ultrafiltered to contain proteins with a molecular weight of 3000 or more.

7. A method for inhibiting stem cell differentiation, characterized by subjecting the residue after hot water extraction of tea leaves to alkaline extraction, adding a differentiation inhibitor containing the resulting protein as an active ingredient to a cell culture medium, and culturing stem cells in the cell culture medium.

8. The method for inhibiting stem cell differentiation described in claim 7, characterized in that after alkaline extraction of the residue, the resulting extract is ultrafiltered, and the protein having a molecular weight of 3000 or more is contained in the differentiation inhibitor.

9. A cell culture medium for stem cell culture, characterized by containing alkaline-extracted protein from tea leaves as an active ingredient.

10. 10. The cell culture medium for stem cell culture according to claim 9, wherein the molecular weight of the alkali-extracted protein is 3,000 or more.

11. 11. The cell culture medium for stem cell culture according to claim 9, wherein the alkali-extracted protein is contained at a concentration of 1 to 400 μg / mL.

12. A method for producing a cell culture medium for stem cell culture, characterized by subjecting tea leaves to a hot water extraction treatment, followed by an alkali extraction treatment of the residue, and incorporating the resulting protein as an active ingredient.

13. The method for producing a cell culture medium for stem cell culture according to claim 12, characterized in that after the residue is subjected to alkaline extraction treatment, the resulting extract is ultrafiltered to contain proteins with a molecular weight of 3000 or more.

14. A method for maintaining stem cells in an undifferentiated state, characterized by subjecting the residue after hot water extraction of tea leaves to alkaline extraction, adding a differentiation inhibitor containing the resulting protein as an active ingredient to a cell culture medium, and culturing stem cells in the cell culture medium.

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