Stem cell differentiation inhibitor, method for producing a stem cell differentiation inhibitor, method for inhibiting stem cell differentiation, cell culture medium for stem cell culture, method for producing a cell culture medium for stem cell culture, and method for maintaining undifferentiated stem cells.

Abstract

The present invention provides a differentiation inhibitor for stem cell culture that can suppress the differentiation of stem cells and increase the cell yield when stem cells are cultured by stirring. [Solution] A differentiation inhibitor for stem cell culture, characterized by containing an alkaline extract of coffee seeds as an active ingredient. Preferably, the coffee seeds are the residue remaining after hot water extraction of coffee seeds. Preferably, the alkaline extract is one from which components with a molecular weight of less than 3000 have been removed.

Description

Technical Field

[0001] The present invention relates to a technique for culturing stem cells, and particularly to a differentiation inhibitor for culturing stem cells and the like.

Background Art

[0002] Conventionally, there has been a problem that it is difficult to proliferate induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), etc. while maintaining good quality. That is, since these stem cells are generally used after being cultured in large quantities in an undifferentiated state and then induced to differentiate into target cells, it is desirable to maintain the undifferentiated state at the stage of proliferation.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0004] However, there are many unclear points in the mechanism for maintaining stem cells in an undifferentiated state, and it has not been easy to culture high-quality stem cells in an undifferentiated state in large quantities or to maintain the obtained high-quality stem cells in an undifferentiated state.

[0005] In addition, when performing agitation culture using a reactor (for example, one with a volume of 30 mL) or the like for culturing stem cells in large quantities, there has been a problem that the cell growth efficiency is inferior to that of adherent culture. Specifically, for example, we were able to increase the number of iPS cells by more than 60 times by adhering them to culture for 4 days, but the cell proliferation rate when the same iPS cells were cultured with agitation was only a few times.

[0006] On the other hand, mass culture of stem cells using adherent culture requires securing a large culture area, which presents a problem in terms of industrial application. Therefore, there was a need to improve the yield of agitated culture in order to mass-produce stem cells industrially.

[0007] Therefore, the inventors diligently conducted research and succeeded in developing a stem cell culture differentiation inhibitor, etc., that can suppress stem cell differentiation and increase the cell yield when stem cells are cultured with agitation, by using an extract obtained by alkaline extraction of coffee seeds as an active ingredient, thereby completing the present invention.

[0008] Here, Patent Document 1 discloses compositions for use in promoting skin wound healing, and specifically describes coffee seed extract as a substance that induces or maintains the expression of COL17A1 in cells. However, this literature described evaluations specifically for skin cells and did not mention the use of extracts obtained by alkaline extraction of coffee seeds.

[0009] Furthermore, Patent Document 2 describes a chilling injury mitigation agent and a necrosis inhibitor containing coffee extract, as well as a method for preserving organs, tissues, or cells of a living organism using a culture medium containing coffee extract. However, this document describes the effects specifically for low-temperature storage, and does not mention normal cultivation, nor does it mention the use of extracts obtained by alkaline extraction of coffee seeds.

[0010] Furthermore, Patent Document 3 discloses a method for reducing the number or percentage of pluripotent stem cells in a cell population by contacting the cell population containing pluripotent stem cells with a pluripotent stem cell selective inhibitor or cytotoxic agent, and it is described that caffeic acid is used as the pluripotent stem cell selective inhibitor or cytotoxic agent. In other words, this document describes the use of coffee extract to inhibit the culture of pluripotent stem cells, but it does not describe the use of extract obtained by alkaline extraction of coffee seeds, nor does it describe how this suppresses stem cell differentiation or increases cell yield when stem cells are cultured with agitation.

[0011] The present invention has been made in view of the above circumstances, and aims to provide a differentiation inhibitor for stem cell culture that can suppress the differentiation of stem cells and increase the cell yield when stem cells are cultured by stirring, a method for producing a differentiation inhibitor for stem cell culture, a method for suppressing the differentiation of stem cells, a cell culture medium for stem cell culture, a method for producing a cell culture medium for stem cell culture, and a method for maintaining undifferentiated stem cells in culture. [Means for solving the problem]

[0012] To achieve the above objective, the stem cell culture differentiation inhibitor of the present invention contains an alkaline extract of coffee seeds 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 coffee seed is the residue after hot water extraction of the coffee seed. Furthermore, it is preferable that the differentiation inhibitor for stem cell culture of the present invention be configured such that the alkaline extract has components with a molecular weight of less than 3000 removed.

[0013] The present invention provides a method for producing a differentiation inhibitor for stem cell culture, which involves subjecting the residue obtained after hot water extraction of coffee seeds to alkaline extraction, and incorporating the resulting extract as an active ingredient. In addition, the method for producing the differentiation inhibitor for stem cell culture of the present invention is preferably a method in which after the alkali extraction treatment of the residue, the obtained extract is ultrafiltered to remove components having a molecular weight of less than 3000 as the extract and the resulting product is contained.

[0014] The method for inhibiting differentiation of stem cells of the present invention is a method in which a residue obtained by subjecting coffee seeds to a hot water extraction treatment is subjected to an alkali extraction treatment, and a differentiation inhibitor containing the obtained extract as an active ingredient is added to a cell culture solution, and the stem cells are cultured with the cell culture solution. In addition, the method for inhibiting differentiation of stem cells of the present invention is preferably a method in which after the alkali extraction treatment of the residue, the obtained extract is ultrafiltered to remove components having a molecular weight of less than 3000 as the extract and the resulting product is contained in the differentiation inhibitor.

[0015] In addition, the method for inhibiting differentiation of stem cells of the present invention is preferably a method in which the protein concentration in the cell culture solution is 1.5 to 40 μg / mL. Furthermore, the method for inhibiting differentiation of stem cells of the present invention is preferably a method in which the stem cells are pluripotent stem cells.

[0016] The cell culture solution for stem cell culture of the present invention is configured to contain an alkali extract of coffee seeds as an active ingredient. In addition, it is also preferable that the cell culture solution for stem cell culture of the present invention is configured such that the alkali extract is one from which components having a molecular weight of less than 3000 have been removed.

[0017] The method for producing the cell culture solution for stem cell culture of the present invention is a method in which a residue obtained by subjecting coffee seeds to a hot water extraction treatment is subjected to an alkali extraction treatment, and the obtained extract is contained as an active ingredient. In addition, the method for producing the cell culture solution for stem cell culture of the present invention is also preferably a method in which after the alkali extraction treatment of the residue, the obtained extract is ultrafiltered to remove components having a molecular weight of less than 3000 as the extract and the resulting product is contained.

[0018] The undifferentiated maintenance culture method of the stem cells of the present invention is a method in which the residue after subjecting coffee seeds to hot water extraction treatment is subjected to alkali extraction treatment, and a differentiation inhibitor containing the obtained extract as an active ingredient is added to a cell culture solution, and the stem cells are cultured with the cell culture solution.

Effects of the Invention

[0019] According to the present invention, it is possible to provide a differentiation inhibitor for stem cell culture, a method for producing a differentiation inhibitor for stem cell culture, a method for inhibiting the differentiation of stem cells, a cell culture solution for stem cell culture, a method for producing a cell culture solution for stem cell culture, and an undifferentiated maintenance culture method of stem cells, which can suppress the differentiation of stem cells and increase the cell yield when the stem cells are cultured by stirring culture.

Brief Description of the Drawings

[0020] [Figure 1] It is a figure which shows the result (without ultrafiltration) of Test 1 for confirming the differentiation inhibitory effect of a differentiation inhibitor etc. for stem cell culture which concerns on embodiment of this invention. [Figure 2] It is a figure which shows the result (with ultrafiltration) of Test 1 for confirming the differentiation inhibitory effect of a differentiation inhibitor etc. for stem cell culture which concerns on embodiment of this invention. [Figure 3] It is a figure which shows the result (without ultrafiltration) of Test 2 for confirming the effect of increasing the yield by stirring culture of cells using a differentiation inhibitor etc. for stem cell culture which concerns on embodiment of this invention. [Figure 4] It is a figure which shows the result (with ultrafiltration) of Test 3 for confirming the effect of increasing the yield by stirring culture of cells using a differentiation inhibitor etc. for stem cell culture which concerns on embodiment of this invention.

Modes for Carrying Out the Invention

[0021] The following describes in detail embodiments of the present invention concerning the differentiation inhibitor for stem cell culture, the method for producing the differentiation inhibitor for stem cell culture, the method for inhibiting the differentiation of stem cells, 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 the undifferentiated state of stem cells. However, the present invention is not limited to the following embodiments and the specific details of the examples described later.

[0022] The stem cell culture differentiation inhibitor of this embodiment, when added to the cell culture medium used for culturing stem cells, provides both a stem cell differentiation inhibitory effect and an increased yield effect through agitation during stem cell culture. The stem cell culture differentiation inhibitor of this embodiment is characterized by containing an alkaline extract of coffee seeds as an active ingredient.

[0023] In the differentiation inhibitor for stem cell culture of this embodiment, the coffee seeds are preferably the residue remaining after hot water extraction of the coffee seeds. Alternatively, the coffee seeds can be those that have been roasted and then subjected to hot water extraction. The hot water extraction can be performed, for example, by the conventional method of dripping coffee, or by stirring in 90°C hot water and removing the hot water by centrifugation.

[0024] It has been reported that the amount of coffee waste in Japan is estimated to be around 800,000 tons per year. The stem cell culture differentiation inhibitor of this embodiment makes it possible to effectively utilize food waste and obtain the above-mentioned excellent effects. However, in the differentiation inhibitor for stem cell culture of this embodiment, it is also possible to use unroasted coffee seeds, untreated coffee seeds, or dried coffee seeds without roasting or hot water extraction.

[0025] In the differentiation inhibitor for stem cell culture of this embodiment, the alkaline extract of coffee seeds can be obtained by immersing and dissolving coffee seeds in an alkaline solution. Specifically, for example, the residue remaining after hot water extraction of coffee seeds is mixed with an aqueous sodium hydroxide solution and stirred with a stirrer for a predetermined time. Then, by centrifuging the resulting composition and collecting the supernatant, an alkaline extract containing alkaline extract proteins from coffee seeds can be obtained.

[0026] In the differentiation inhibitor for stem cell culture of this embodiment, it is preferable that the alkaline extract is one from which components with a molecular weight of less than 3000 have been removed. In other words, it is preferable to ultrafilter the alkaline extract of coffee seeds to ensure that the differentiation inhibitor for stem cell culture contains only components with a molecular weight of 3000 or more.

[0027] As mentioned above, caffeic acid contained in coffee seeds has been reported to inhibit stem cell culture. As shown in the examples described later, even with the stem cell culture differentiation inhibitor of this embodiment, which contains a component with a molecular weight of less than 3000 as an alkaline extract, it is possible to obtain an excellent stem cell differentiation inhibitory effect and an increased yield effect by stirring the culture of stem cells. On the other hand, a differentiation inhibitor for stem cell culture obtained by ultrafiltration of coffee seed extract to remove components with a molecular weight of less than 3000 can further improve the effect of inhibiting stem cell differentiation and increasing the yield of stem cells through agitated culture compared to an inhibitor containing components with a molecular weight of less than 3000.

[0028] The stem cell culture differentiation inhibitor of this embodiment can be used by adding it to a cell culture medium to adjust the final concentration of the alkaline extract protein to a predetermined range, as described later.

[0029] As stem cells cultured using the differentiation inhibitor for stem cell culture of this embodiment, induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells) can be suitably used. The same applies to the stem cells in the method for producing a differentiation inhibitor for stem cell culture, the method for inhibiting the differentiation of stem cells, 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 this embodiment.

[0030] The method for producing a differentiation inhibitor for stem cell culture according to this embodiment is characterized by subjecting the residue obtained after hot water extraction of coffee seeds to alkaline extraction treatment, and including the resulting extract as an active ingredient. Furthermore, in the method for producing a differentiation inhibitor for stem cell culture according to this embodiment, it is preferable to include an extract obtained by ultrafiltration of the residue after hot water extraction of coffee seeds, from which components with a molecular weight of less than 3000 have been removed. According to the method for producing a differentiation inhibitor for stem cell culture of this embodiment, it is possible to suitably obtain a differentiation inhibitor for stem cell culture that has a superior effect in inhibiting stem cell differentiation and increasing yield through agitated culture.

[0031] The stem cell differentiation inhibition method of this embodiment is characterized by subjecting the residue obtained after hot water extraction of coffee seeds to alkaline extraction, adding a differentiation inhibitor containing the obtained extract as an active ingredient to a cell culture medium, and culturing stem cells in the cell culture medium. Furthermore, in the stem cell differentiation inhibition method of this embodiment, it is preferable to include in the differentiation inhibitor an extract obtained by ultrafiltration of the residue after hot water extraction of coffee seeds, and then removing components with a molecular weight of less than 3000 from the resulting extract.

[0032] Furthermore, in the stem cell differentiation suppression method of this embodiment, the concentration of protein derived from the alkaline extract of coffee seeds contained in the cell culture medium is preferably 1 to 40 μg / mL, more preferably 1 to 30 μg / mL, even more preferably 1.3 to 25 μg / mL, and particularly preferably 1.5 to 20 μg / mL. According to this embodiment of the method for suppressing stem cell differentiation, it is possible to suitably suppress stem cell differentiation during stem cell culture.

[0033] The cell culture medium for stem cell culture according to this embodiment is characterized by containing an alkaline extract of coffee seeds as an active ingredient. Furthermore, it is preferable that the cell culture medium for stem cell culture in this embodiment is an alkaline extract of coffee seeds from which components with a molecular weight of less than 3000 have been removed.

[0034] Furthermore, the cell culture medium for stem cell culture in this embodiment preferably has a concentration of protein derived from the alkaline extract of coffee seeds of 1 to 40 μg / mL, more preferably 1 to 30 μg / mL, even more preferably 1.3 to 25 μg / mL, and particularly preferably 1.5 to 20 μg / mL. According to the stem cell culture medium of this embodiment, it is possible to obtain an excellent effect of inhibiting stem cell differentiation and an increase in yield through agitation culture when culturing stem cells.

[0035] The method for producing a cell culture medium for stem cell culture according to this embodiment is characterized by subjecting the residue obtained after hot water extraction of coffee seeds to alkaline extraction treatment, and including the resulting extract as an active ingredient. Furthermore, in the method for producing a cell culture medium for stem cell culture according to this embodiment, it is preferable to include an extract obtained by ultrafiltration of the residue after hot water extraction of coffee seeds, thereby removing components with a molecular weight of less than 3000.

[0036] Furthermore, in the method for producing a cell culture medium for stem cell culture according to this embodiment, the concentration of protein derived from the alkaline extract of coffee seeds is preferably 1 to 40 μg / mL, more preferably 1 to 30 μg / mL, even more preferably 1.3 to 25 μg / mL, and particularly preferably 1.5 to 20 μg / mL. According to the method for producing a cell culture medium for stem cell culture in this embodiment, it is possible to suitably obtain a cell culture medium for stem cell culture that has both a stem cell differentiation inhibitory effect and a yield-increasing effect due to agitated culture.

[0037] The method for maintaining undifferentiated stem cells according to this embodiment is characterized by subjecting the residue obtained after hot water extraction of coffee seeds to alkaline extraction, adding a differentiation inhibitor containing the obtained extract as an active ingredient to a cell culture medium, and culturing stem cells in the cell culture medium. According to this embodiment of the method for maintaining the undifferentiated state of stem cells during culture, it is possible to suitably culture stem cells while maintaining their undifferentiated state. [Examples]

[0038] [Test 1] Tests were conducted as follows to confirm the differentiation-inhibiting effect of the stem cell culture differentiation inhibitor, etc., according to the embodiment of the present invention.

[0039] <Removal of water-soluble components derived from coffee> Coffee seeds were used as a raw material for a differentiation inhibitor for stem cell culture, and water-soluble components derived from coffee were removed from the coffee seeds by washing them with hot water. Specifically, 1g of coffee seed powder (commercially available drip coffee powder "A Little Luxurious Coffee Shop® Premium Drip Aromatic Deep Roast Kilimanjaro Blend," Ajinomoto AGF Co., Ltd.) and 50mL of ultrapure water were mixed and stirred at 90°C for 3 hours using a hot stirrer (AS ONE Corporation). Next, the obtained solution was centrifuged (4500 × g, 60 minutes) and the supernatant was removed. Furthermore, the same hot water washing procedure was repeated two more times (a total of three times) to obtain coffee seed powder from which water-soluble components had been removed.

[0040] <Alkaline extraction of coffee seeds> The entire amount of coffee seed powder obtained by the above procedure was mixed with 50 mL of a 0.05 M aqueous solution of NaOH (Fujifilm Wako Pure Chemical Industries, Ltd.) and stirred at 60°C for 3 hours using a hot stirrer (AS ONE Corporation). Next, the obtained composition was centrifuged (11000 × g, 60 minutes), and the supernatant was collected to obtain Sample 1, an alkaline extract of coffee seeds containing alkali-soluble components (hereinafter sometimes referred to as coffee extract).

[0041] <Preparation of components with a molecular weight cutoff of 3000 or more by ultrafiltration> Next, sample 1 of the coffee extract was ultrafiltered to remove components with a molecular weight of 3000 or more, while removing components with a molecular weight of less than 3000. Specifically, sample 1 of coffee extract was placed in an ultrafiltration filter (NanoSep®, molecular weight cutoff 3000, Cytiva) at a rate of 500 μL / container, and centrifuged (11000 × g, 25 minutes) to remove low molecular weight components.

[0042] At this point, an equal volume of phosphate buffer (Nacalai Tesque Co., Ltd.) was added to the high molecular weight component side to replace the volume of liquid transferred to the low molecular weight fraction, and centrifugation was repeated seven times. Then, 150 μL of the obtained high molecular weight component liquid was mixed with 390 μL of phosphate buffer to obtain Sample 2 of the coffee extract from which the low molecular weight component had been removed. In sample 2 of the coffee extract, the calculated residual percentage of low molecular weight components was 0.00018% before ultrafiltration, which was sufficiently small to be negligible.

[0043] <Measurement of protein concentration in coffee extract> The protein concentrations of coffee extract samples 1 and 2 were measured using the BCA method. Specifically, a protein concentration measurement kit (TaKaRa BCA Protein Assay Kit, Takara Bio Inc.) was used. Following the instructions, the prepared BCA reaction solution and the sample to be measured, which had been appropriately diluted with phosphate buffer (Nacalai Tesque Corporation), were mixed in equal volumes in a 96-well plate (351172, Corning). After standing at 37°C for 2 hours, the absorbance was measured using a microplate reader (Corona Electric Corporation) at a primary wavelength of 562 nm and a secondary wavelength of 447 nm to determine the protein concentration. Bovine serum albumin (BSA) included in the kit was used as a calibration curve. The protein concentrations of coffee extract samples 1 and 2 were 2.2 mg / mL and 5.0 mg / mL, respectively.

[0044] <Cell adhesion culture> Next, cell adhesion culture was performed using coffee extract samples 1 and 2. Specifically, iPS cells (1231A3 strain, Kyoto University) were used as the cells, and the cells were suspended in a culture medium (StemFit, Ajinomoto Co., Inc.) containing 10 mM Y-27632 (Fujifilm Wako Pure Chemical Corporation) and 15.7 mg / mL iMatrix511 (Nippi Corporation), and then placed in five 48-well plates (cell adhesion treated, Corning) in a 1.5 × 10⁶ arrangement. 3 Seeds were sown at 300 μL / well, and the medium was replaced the day after sowing with one that did not contain Y-27632 or iMatrix-511.

[0045] Next, coffee extract samples 1 and 2 were added to the culture medium, and cell culture was performed. The coffee extract was added between day 2 and day 7 after the start of culture, and the cell culture was carried out with the coffee extract added for a total of 6 days.

[0046] In this case, for coffee extract sample 1, cell culture was performed by adding it to the culture medium so that the final concentrations of coffee seed alkaline extract-derived protein (hereinafter sometimes referred to as coffee-derived protein) were 1.76 μg / mL (Example 1), 8.8 μg / mL (Example 2), and 44 μg / mL (Comparative Example 2), respectively. Furthermore, for coffee extract sample 2, cell culture was performed by adding it to the culture medium so that the final concentrations of coffee-derived protein were 4.0 μg / mL (Example 3), 20.0 μg / mL (Example 4), and 100 μg / mL (Comparative Example 3), respectively. Furthermore, cell culture was performed using a medium with a final concentration of coffee-derived protein of 0 μg / mL without adding coffee extract (Comparative Example 1).

[0047] <Evaluation of the inhibitory effect on cell differentiation> The differentiation-inhibiting effect of coffee extract was evaluated using cells obtained from the cell culture described above. Specifically, at the end of the culture period on day 7 after the start of culture, 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 Co., Ltd.).

[0048] Then, the differentiation-inhibiting effect of coffee extract samples 1 and 2, respectively, on cells cultured in a culture medium was evaluated using flow cytometry and undifferentiated markers. Specifically, cells were stained using an antibody (Alexa Flour488(R) labeled anti-SSEA-1 antibody, R&D Systems), and fluorescence intensity was measured using a flow cytometer (CytoFLEX S, Beckman Coulter). Cells not stained with the antibody were used as a reference for determining positive / negative results. The results are shown in Figures 1 and 2. In this evaluation, a lower positive rate indicates that the cells are undifferentiated.

[0049] As shown in Figure 1, regarding the inhibitory effect of coffee extract sample 1 (without ultrafiltration) on cell differentiation, in Comparative Example 1 with a coffee-derived protein concentration of 0 μg / mL, the SSEA-1 positive rate was 21.34%; in Example 1 with a coffee-derived protein concentration of 1.76 μg / mL, the SSEA-1 positive rate was 11.41%; in Example 2 with a coffee-derived protein concentration of 8.8 μg / mL, the SSEA-1 positive rate was 11.72%; and in Comparative Example 2 with a coffee-derived protein concentration of 44 μg / mL, the SSEA-1 positive rate was 31.62%.

[0050] Furthermore, as shown in Figure 2, regarding the inhibitory effect of coffee extract sample 2 (with ultrafiltration) on cell differentiation, in Comparative Example 1 with a coffee-derived protein concentration of 0 μg / mL, the SSEA-1 positive rate was 21.34%, in Example 3 with a coffee-derived protein concentration of 4.0 μg / mL, the SSEA-1 positive rate was 7.82%, in Example 4 with a coffee-derived protein concentration of 20.0 μg / mL, the SSEA-1 positive rate was 12.42%, and in Comparative Example 3 with a coffee-derived protein concentration of 100 μg / mL, the SSEA-1 positive rate was 54.77%.

[0051] In other words, in Examples 1 to 4, where coffee-derived protein was added to a concentration in the range of 1.76 μg / mL to 20.0 μg / mL, the positive rate was lower compared to Comparative Example 1, in which no coffee extract was added. On the other hand, in Comparative Examples 2 and 3, where coffee-derived protein was added at a concentration of 44 μg / mL or higher, the positive rate was higher compared to Comparative Example 1, where no coffee extract was added. These results demonstrate that the stem cell culture differentiation inhibitor of this embodiment, when prepared and used in a predetermined concentration range in the cell culture medium, can effectively suppress the phenomenon of uncontrolled differentiation of iPS cells and improve the quality of iPS cell culture.

[0052] [Exam 2] The following tests were conducted to confirm the yield-increasing effect of stirring cell culture using a differentiation inhibitor for stem cell culture according to an embodiment of the present invention.

[0053] <Cell culture using coffee extract-containing medium, part 1: Stirring Cells were cultured using agitation in a culture medium containing Sample 1 of the coffee extract from Test 1 (without ultrafiltration) and in a culture medium that did not contain the coffee extract. Specifically, iPS cells (1231A3 strain, Kyoto University) cultured on a plate were suspended in a culture medium (StemFit, Ajinomoto Co., Inc.) containing 10 mM Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.), and then 1.5 × 10¹⁶ cells were placed in each of two 30 mL culture reactors (Able Co., Ltd.). 6 Seeds were sown at a rate of 1 per reactor, or 30 mL per reactor.

[0054] Cell cultures were performed in two cases: one where 70% of the culture medium was replaced with a medium that did not contain Y-27632 the day after sowing, and coffee-derived protein was added to the medium so that the final concentration was 1.76 μg / mL (Example 5); and another where no coffee extract was added to the culture medium (Comparative Example 4). After culturing for another 3 days, the aggregated cells were dissociated into individual cells using a detachment solution (TrypLE Select CTS) diluted 2-fold with phosphate buffer containing 0.5 mM EDTA, and the number of viable cells was measured using a cell counter (NucleoCounter(R) NC-200(TM), Chemometec). The results are shown in Figure 3.

[0055] As shown in Figure 3, in Comparative Example 4, where the concentration of coffee-derived protein was 0 μg / mL, the number of viable cells was 2.412 × 10⁶. 7 The proliferation rate was 1.61 times. In contrast, in Example 5, where the coffee-derived protein concentration was 1.76 μg / mL, the number of viable cells was 3.210 × 10⁶. 7 The proliferation rate was 2.14 times.

[0056] These results clearly show that, according to sample 1 of coffee extract (without ultrafiltration), a yield increase effect can be obtained by stirring the cells during culture. Furthermore, this result means that less culture medium is needed to obtain the desired number of cells, confirming that the differentiation inhibitor for stem cell culture in this embodiment is industrially useful.

[0057] [Exam 3] The following tests were conducted to confirm the yield-increasing effect of stirring cell culture using a differentiation inhibitor for stem cell culture according to an embodiment of the present invention.

[0058] <Cell culture using coffee extract-containing medium, part 2> Cells were stirred and cultured using a culture medium containing Sample 2 (ultrafiltration) of the coffee extract from Test 1. Specifically, iPS cells (1231A3 strain, Kyoto University) cultured on a plate were suspended in a culture medium (StemFit, Ajinomoto Co., Inc.) containing 10 mM Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.), and then 1.5 × 10¹⁶ cells were placed in each 30 mL culture reactor (Able Co., Ltd.). 6 Seeds were sown at a rate of 1 per reactor, or 30 mL per reactor.

[0059] Cell cultures were performed using two methods: one in which 70% of the culture medium was replaced with a medium that did not contain Y-27632 the day after sowing, and coffee-derived protein was added to the medium so that the final concentration was 4.0 μg / mL (Example 6); and another in which no coffee extract was added to the culture medium (Comparative Example 5). After culturing for another 3 days, the aggregated cells were dissociated into individual cells using a detachment solution (TrypLE Select CTS) diluted 2-fold with phosphate buffer containing 0.5 mM EDTA, and the number of viable cells was measured using a cell counter (NucleoCounter(R) NC-200(TM), Chemometec). The results are shown in Figure 4.

[0060] As shown in Figure 4, in Comparative Example 5, where the concentration of coffee-derived protein was 0 μg / mL, the number of viable cells was 5.124 × 10⁶. 7 The proliferation rate was 3.41 times. In contrast, in Example 6, where the coffee-derived protein concentration was 4.0 μg / mL, the number of viable cells was 8.724 × 10⁶. 7 The proliferation rate was 5.81 times.

[0061] Specifically, while Example 5, which used a coffee extract containing components with a molecular weight of less than 3000 without ultrafiltration as in Test 2, had a growth ratio of 2.14, Example 6, which used a coffee extract from which components with a molecular weight of less than 3000 were removed by ultrafiltration as in Test 3, had a growth ratio of 5.81. This indicates that the differentiation inhibitor for stem cell culture obtained by ultrafiltration has an even better effect on increasing the yield of cells through agitation culture than the differentiation inhibitor for stem cell culture obtained without ultrafiltration.

[0062] The present invention is not limited to the embodiments and examples described above, and it goes without saying that various modifications can be made within the scope of the present invention. For example, it is possible to make appropriate modifications, such as using coffee seeds that have not undergone hot water extraction treatment. [Industrial applicability]

[0063] This invention can be suitably used, for example, to produce a stem cell culture differentiation inhibitor that has excellent effects in inhibiting stem cell differentiation and increasing yield through agitated culture, by effectively utilizing the residue after hot water extraction of coffee seeds, which is often considered waste.

Claims

1. A differentiation inhibitor for stem cell culture, characterized by containing an alkaline extract of coffee seeds as an active ingredient.

2. The differentiation inhibitor for stem cell culture according to claim 1, characterized in that the coffee seeds are the residue after hot water extraction of the coffee seeds.

3. The differentiation inhibitor for stem cell culture according to claim 1 or 2, characterized in that the alkaline extract is obtained by removing components with a molecular weight of less than 3000.

4. A method for producing a differentiation inhibitor for stem cell culture, characterized by subjecting the residue obtained after hot water extraction of coffee seeds to alkaline extraction, and including the resulting extract as an active ingredient.

5. The method for producing a differentiation inhibitor for stem cell culture according to claim 4, characterized in that, after alkaline extraction treatment of the residue, the obtained extract is ultrafiltered, and the extract contains components with a molecular weight of less than 3000 removed.

6. A method for inhibiting stem cell differentiation, characterized by subjecting the residue obtained after hot water extraction of coffee seeds to alkaline extraction, adding a differentiation inhibitor containing the obtained extract as an active ingredient to a cell culture medium, and culturing stem cells in the cell culture medium.

7. The method for inhibiting stem cell differentiation according to claim 6, characterized in that, after alkaline extraction of the residue, the obtained extract is ultrafiltered, and the extract from which components with a molecular weight of less than 3000 have been removed is included in the differentiation inhibitor.

8. The method for suppressing stem cell differentiation according to claim 6 or 7, characterized in that the protein concentration of the cell culture medium is 1.5 to 40 μg / mL.

9. The method for suppressing the differentiation of stem cells according to claim 6 or 7, characterized in that the stem cells are pluripotent stem cells.

10. A cell culture medium for stem cell culture, characterized by containing an alkaline extract of coffee seeds as an active ingredient.

11. The cell culture medium for stem cell culture according to claim 10, characterized in that the alkaline extract is obtained by removing components with a molecular weight of less than 3000.

12. A method for producing a cell culture medium for stem cell culture, characterized by subjecting the residue obtained after hot water extraction of coffee seeds to alkaline extraction, and including the resulting extract 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 alkaline extraction treatment of the residue, the obtained extract is ultrafiltered to remove components with a molecular weight of less than 3000 from the extract.

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

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