JP2013247927A - Rice bran-originated cell proliferation promoter - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide, by finding out a plant-originated component having a proliferation-promoting effect toward cultured animal cells, a cell proliferation promoter, an additive for a medium, and the medium for culturing the cells, containing the component.SOLUTION: Since the rice bran extract originated from a plant has an excellent cell proliferation-promoting effect and has a high safety to human bodies, it is useful for the active ingredient of a cell proliferation promoter and an additive for a medium. The cells cultured in the medium containing the rice bran extract can be applied to the production of protein pharmaceuticals, regenerative medicine and cell therapy.

Description

  The present invention relates to a cell growth promoter derived from rice bran, a medium additive containing the cell growth promoter, and a cell culture medium.

  In recent years, it has become important in the field of life science to produce useful substances on an industrial scale by culturing a large amount of cells that produce a target substance using cell culture technology. For example, antibody drugs and proteinaceous biopharmaceuticals are produced by mammalian cell culture because of so-called post-translational modifications. In the field of cell therapy such as regenerative medicine for cultured skin and adoptive immunotherapy for cancer, the cells themselves are used as products or as part of products. In particular, stem cell technology uses cells and tissues that have been induced to differentiate from a certain stem cell. In these fields, isolated useful cells are amplified, induced for differentiation, activated outside the body, and further processed into products. In cell culture carried out for such a purpose, it is natural that the culture efficiency is high, and safety without concern about infection is emphasized above all.

  In general, when culturing animal cells, a culture medium in which a cell growth factor is added to a basal medium composed of amino acids, vitamins, inorganic salts, sugars and the like is used. As cell growth factors, animal-derived factors such as fetal calf serum, calf serum, bovine serum albumin, and transferrin are usually used. However, it is said that the use of animal-derived factors should be avoided because there are concerns about zoonotic diseases such as BSE. On the other hand, there are still problems to be solved, such as the high safety of serum-free medium and protein-free medium, in which cell proliferation ability is inferior, and cell death is likely to occur due to various stresses.

Sericin is known as an animal cell growth factor not derived from mammals (see, for example, Patent Documents 1 and 2). Sericin is derived from the silkworm, silkworm, and silk thread has long been used as a surgical thread. Therefore, it is considered to be highly safe for the human body. Patent Document 3 discloses fructan as a plant-derived animal cell growth factor.

International Publication WO02 / 86133 JP 2011-136852 A JP 2008-228587 A

  An object of the present invention is to find a plant-derived component having a growth promoting action on cultured animal cells, and to provide a cell growth promoting agent, a medium additive and a cell culture medium containing the component.

The present invention includes the following inventions in order to solve the above problems.
[1] A cell growth promoter comprising rice bran extract as an active ingredient.
[2] The cell growth promoter according to [1], wherein the rice bran extract contains 50% by mass or more of protein.
[3] A medium additive comprising the cell growth promoter according to [1] or [2].
[4] A cell culture medium comprising the cell growth promoter according to [1] or [2].
[5] The medium according to [4], wherein the cells are animal cells.
[6] The medium according to the above [5], wherein the animal cell is a mammalian cell.
[7] The medium according to [6], wherein the mammalian cell is a hybridoma, a recombinant protein-producing cell, or a stem cell.

  ADVANTAGE OF THE INVENTION According to this invention, the cell growth promoter which uses a rice bran extract as an active ingredient can be provided. Since rice bran extract is derived from plants and has high safety to the human body, cells cultured in a medium containing the cell growth promoter can be applied to protein drug production, regenerative medicine, and cell therapy.

It is a figure which shows the result of having examined the effect with respect to the cell proliferation of a mouse hybridoma about the rice bran extract (RBP55) of protein content 55%. It is a figure which shows the result of having examined the optimal density | concentration of the rice bran extract (RBP55) with a protein content of 55% regarding the cell growth promotion effect with respect to a mouse hybridoma. It is a figure which shows the result of having compared the cell growth promotion effect with respect to the mouse hybridoma of the rice bran extract (RBP55) of protein content 55% with the cell growth promotion effect of bovine serum albumin (BSA). It is a figure which shows the result of having examined the optimal density | concentration of the rice bran extract (RBP74) of protein content 74% regarding the cell growth promotion effect with respect to a mouse hybridoma. It is a figure which shows the result of having examined the effect with respect to the monoclonal antibody production with respect to a mouse | mouth hybridoma of the rice bran extract (RBP55) of protein content 55%. It is a figure which shows the result of having examined the effect with respect to the cell proliferation of a mouse | mouth hybridoma about the rice bran extract (RBP74) of protein content 74% of molecular weight fractionation. It is a figure which shows the result of having examined the effect with respect to the cell proliferation of a mouse hybridoma about what carried out the high pressure steam sterilization processing of the rice bran extract (RBP74) of protein content 74% by which molecular weight fractionation was carried out. It is a figure which shows the result of having examined the effect with respect to the cell growth of a CHO cell about the rice bran extract (RBP55) of protein content 55%. It is a figure which shows the result of having examined the effect with respect to the recombinant protein production with respect to the CHO cell of the rice bran extract (RBP55) with a protein content of 55%. It is a figure which shows the result of having examined the effect with respect to the cell growth of HepG2 cell about the rice bran extract (RBP55) of protein content 55%. It is a figure which shows the result of having examined the effect with respect to the differentiation ability of a mesenchymal stem cell about the rice bran extract (RBP55) of protein content 55%.

  The cell growth promoter of the present invention only needs to contain rice bran extract as an active ingredient. Rice bran, which is a raw material for obtaining a rice bran extract, can be obtained by milling brown rice. The rice bran preferably has a low white rice content. The rice bran is preferably a rice bran obtained from milled rice having a rice milling ratio of 85% or more, more preferably a rice bran obtained from milled rice having a rice milling ratio of 90% or more, and more preferably a rice bran obtained from milled rice having a rice milling ratio of 95% or more. It is. The rice bran contains defatted rice bran, and the defatted rice bran, which is a residue obtained by extracting rice oil from the rice bran, can be suitably used as a raw material for the rice bran extract in the present invention.

  The rice bran extract preferably contains a rice bran-derived protein (hereinafter referred to as “rice bran protein”). Such a rice bran extract can be obtained by extracting rice bran as a raw material with water or an organic solvent. Examples of the solvent used for extraction include water (for example, purified water, distilled water, tap water, etc.), salt (for example, sodium chloride, etc.) solution, neutral to weak alkaline solution that can be used for food material preparation, food Examples thereof include organic solvents that can be used for preparing the material (for example, ethanol), solvents that are appropriately combined with these, and the like. A dilute salt solution, a neutral to weak alkaline solution, or a neutral to weak alkaline solution containing about 5 to 90% by volume of ethanol, more preferably a neutral to weak alkaline solution. The pH of the neutral to weak alkaline solution is preferably about 6.5 to 12, more preferably about 6.5 to 11, more preferably about 7.0 to 11, still more preferably about 7.5 to 10, and about 7 .5-9 are particularly preferred. Examples of the alkali include sodium hydroxide and potassium hydroxide. The neutral to weak alkaline solution is preferably an aqueous solution.

  The extraction method is not particularly limited, and can be appropriately selected from known extraction methods. For example, rice bran and a solvent are mixed to obtain a mixed solution, and the rice bran and the extract are separated after a certain time. The obtained extract may be used as a rice bran extract as it is, or may be used after the extract is concentrated by a known method.

  The blending ratio of rice bran and solvent is not particularly limited, but is usually about 5 to 50 parts by mass of solvent with respect to 1 part by mass of rice bran, preferably about 5 to 20 parts by mass of solvent with respect to 1 part by mass of rice bran, and more preferably Is about 8-15 parts by weight of solvent per 1 part by weight of rice bran. When extracting rice bran protein with, for example, a neutral or weakly alkaline aqueous solution, the rice bran and the neutral or weakly alkaline aqueous solution adjusted to the above pH may be mixed at the above blending ratio, and the rice bran and water are mixed at the above blending ratio. Then, a sodium hydroxide aqueous solution or a potassium hydroxide aqueous solution having a concentration of about 10 to 30% by mass may be added to adjust the pH. The mixing of the rice bran and the solvent is preferably performed with stirring, shaking, etc. in order to mix the rice bran uniformly.

A method for separating rice bran and rice bran extract from a mixed solution of rice bran and solvent is not particularly limited, and examples thereof include a centrifugal separation method, a filtration method, a vacuum membrane filtration method, a pressure membrane filtration method, and a stationary decant method. . A centrifugal separation method is preferable. The obtained rice bran extract contains rice bran protein, dietary fiber, carbohydrates and the like. Although the rice bran extract can be used as it is as an active ingredient of the cell growth promoter of the present invention, the protein concentration can be determined by using, for example, acid precipitation, alcohol precipitation, salting out, membrane separation, chromatographic separation and the like. Is preferably increased.

  For acid precipitation, an inorganic acid such as hydrochloric acid or sulfuric acid; or an organic acid such as trichloroacetic acid or sulfosalicylic acid is added to the rice bran extract to adjust the pH of the extract to slightly acidic, and rice bran protein is precipitated. And a method for separating and obtaining the precipitate. The pH by acid addition is preferably about 2.5 to 5.5. Examples of the alcohol precipitation include a method in which ethanol is added to a rice bran extract so as to be about 50 to 70% by volume to precipitate rice bran protein, and the precipitate is separated and acquired. Similarly to alcohol precipitation, for example, rice bran protein may be precipitated by adding an organic solvent such as acetone instead of alcohol. Examples of salting out include a method of precipitating rice bran protein by adding ammonium sulfate or the like to the rice bran extract. The acid precipitation method is preferred.

  The obtained precipitate is a rice bran extract having a high rice bran protein content, and can be used as it is as an active ingredient of the cell growth promoter of the present invention. Further, by washing the obtained precipitate with an alcohol, an acidic solution, an aqueous solution containing various salts, etc., carbohydrates and phytin can be removed, and a rice bran extract containing a higher concentration of rice bran protein can be obtained. Furthermore, it can be stored frozen and thawed for use as a rice bran extract. Moreover, the obtained precipitation can be pulverized by a well-known drying method, and a preservability can be improved.

  Examples of the method for separating the precipitate include a centrifugal separation method, a membrane filtration method, and a filtration method using a filter aid. Industrially, it is preferable to perform precipitation fractionation by performing a batch-type or continuous-type centrifugal separation using a screw decanter, a basket-type centrifuge, a shear press-type centrifuge, a disk-type centrifuge, or the like. When the precipitate is separated by a membrane filtration method, it can be separated by a pressure difference using a cellulose membrane, an ultrafiltration membrane or the like. It can also be filtered off using a filter aid such as Celite. Examples of the drying method include natural drying, heat drying, reduced pressure room temperature drying, vacuum drying, freeze drying, spray drying and the like. Preferably, freeze drying or spray drying is used.

  Examples of the membrane separation method include a method of concentrating rice bran protein contained in the rice bran extract with an ultrafiltration membrane or the like. Examples of chromatographic separation methods include molecular sieve chromatography and ion exchange column chromatography.

  The rice bran extract, which is an active ingredient of the cell growth promoter of the present invention, may be one obtained by molecular weight fractionation. The method of molecular weight fractionation is not particularly limited, and can be performed using a known method. For example, a commercially available ultrafiltration membrane, gel filtration chromatography or the like can be suitably used. When the rice bran extract after molecular weight fractionation is used as an active ingredient of a cell growth promoter, it is preferable to use a fraction of about 3 kDa or less or a fraction of about 10 kDa or more.

The protein content in the rice bran extract that is an active ingredient of the cell growth promoter of the present invention is not particularly limited, but the amount of protein by the Kjeldahl method with respect to the rice bran extract (dry mass) is preferably about 50% by mass or more, 60 mass% or more is more preferable, and about 70 mass% or more is further more preferable.
The rice bran extract which is an active ingredient of the cell growth promoter of the present invention can be heat-treated. The heat treatment includes autoclave sterilization conditions.

  The cell growth promoting agent of the present invention may be composed only of a rice bran extract or may contain components other than the rice bran extract. Components other than the rice bran extract are not particularly limited as long as they do not adversely affect cell growth. For example, proteins, saccharides, nucleic acids, lipids, inorganic salts and the like can be mentioned.

  Since the cell growth promoting agent of the present invention has a cell growth promoting action, it can be suitably used as a medium additive for addition to a cell culture medium. The medium additive of the present invention may be in any form as long as it can be added to the medium, dissolved and used. For example, it can be carried out in the form of liquid, powder, granule, tablet and the like. The medium used by adding the medium additive of the present invention is not particularly limited as long as it is a medium for cell culture. By adding the medium additive of the present invention to a known basal medium or complete medium, growth of cells cultured using these mediums can be promoted. Examples of the basal medium include Eagle MEM medium, αMEM medium, DMEM medium, ham medium, RPMI 1640 medium, Fischer's medium, and mixed media thereof. Examples of the complete medium include a serum-free medium, a stem cell culture medium, and a pluripotent stem cell culture medium.

  In addition, a cell culture medium prepared by adding the cell growth promoter of the present invention to a basal medium in advance is also included in the present invention. That is, the present invention provides a cell culture medium containing the cell growth promoter of the present invention. The medium of the present invention contains components such as proteins and polypeptides, hormones, amino acids, vitamins, lipid factors, saccharides, osmotic pressure regulators, pH buffering agents, and surfactants that are usually used in cell culture media. It may be included.

  The cells to be cultured are not particularly limited. Examples of the cell include bacteria, yeast, plant cells, animal cells and the like. Of these, animal cells are preferred. Examples of animal cells include mammals including humans (human, monkey, mouse, rat, hamster, guinea pig, rabbit, cat, dog, sheep, pig, cow, horse, goat, etc.), birds (chicken, ostrich, etc.) , Vertebrates such as reptiles (such as crocodiles), amphibians (such as frogs), fish (such as zebrafish and medaka), and cells such as invertebrates such as insects (such as silkworms, moths, and fruit flies). More preferably, it is a mammalian cell.

  The inventors have shown that the use of the medium of the present invention increases the production of monoclonal antibodies in hybridomas, increases the production of recombinant proteins in recombinant protein-producing cells, and maintains the differentiation ability of stem cells. Thus, the medium of the present invention is preferably used for culturing hybridomas, recombinant protein-producing cells and stem cells, in particular. Stem cells mean cells with self-replicating ability and pluripotency, and include somatic stem cells and pluripotent stem cells. Examples of somatic stem cells include neural stem cells, mesenchymal stem cells, hematopoietic stem cells, and pluripotent stem cells include ES cells (embryonic stem cells) and iPS cells in addition to mesenchymal stem cells and hematopoietic stem cells. (Artificial pluripotent stem cells).

  If the medium of the present invention is used as a medium for cryopreservation of cells, cell death during cryopreservation and cell death associated with freezing operations can be suppressed, and the cell survival rate can be increased. That is, the culture medium additive of the present invention can be used as a cryoprotectant. When the medium of the present invention is used as a cryopreservation medium, it is preferable to add 5 to 20% by volume of DMSO or glycerin.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these.

[Example 1: Preparation of rice bran extract]
30 L of water was added to 3 kg of defatted rice bran, and the mixture was stirred at room temperature for 1 hour while adjusting the pH to 8.5 with a 25 W / V% aqueous sodium hydroxide solution. Then, it isolate | separated into the extract and the residue by centrifugation (1,500 rpm * 30 second). 60% by volume sulfuric acid was added to the separated extract to adjust to pH 3.0, followed by stirring at room temperature for 1 hour. Thereafter, the precipitate was separated by centrifugation (3,000 rpm × 1 minute), and the acid-precipitated (separated) protein was recovered and dried by freeze drying. The yield of the dried product was 230 g, and the yield was 7.7%. The protein content by Kjeldahl method was 55%, and a rice bran extract containing a high concentration of rice bran protein was obtained. The obtained rice bran extract with a protein content of 55% is referred to as “RBP55”.

  10 g of “RBP55” was suspended in 60 mL of 60% by volume aqueous ethanol and stirred for 5 minutes. After stirring, the precipitate was recovered by centrifugation (3,000 rpm × 10 minutes). The precipitate was further suspended in 300 mL of 99.5% ethanol, stirred for 5 minutes, collected by centrifugation (3,000 rpm × 10 minutes), and dried by freeze drying. The yield of the rice bran extract containing the obtained purified rice bran protein was 8.2 g, and the yield was 82% (converted to defatted rice bran: 6.3%). The protein content by Kjeldahl method was 75.0%. The obtained rice bran extract with a protein content of 74% is hereinafter referred to as “RBP74”.

[Example 2: Examination of effects on mouse hybridoma]
The cells include mouse hybridoma cell line 2E3-O (F. Makishima, S. Terada, T. Mikami and E. Suzuki “Interleukin-6 is Antiproliferative to a Mouse Hybridoma Cell Line and Promotive for its Antibody Productivity” Cytotechnology 10. 15- 23. 1992) and ASF104 medium (Ajinomoto Co., Inc., serum-free medium) was used as the medium. Cells were seeded in a 24-well plate at 1 × 10 ⁴ cells / mL / well and cultured for several days according to a conventional method, and then the number of cells was counted.

(2-1) Effect of RBP55 on cell proliferation Medium RBP55 was added so that the concentration was 0.1, 0.3, 1, 3, and 10 g / L. As a negative control (NC), an RBP55-free group was provided, and as a positive control, a sericin (1 g / L) group was provided. In each group, n = 5. In each group, the number of cells was counted on the third day after seeding.
The results are shown in FIG. As is clear from FIG. 1, RBP55 showed cytotoxicity at a concentration of 1 g / L or more, and the cells were almost killed. However, when 0.1 g / L was added, a strong growth-promoting effect was observed, and the effect greatly exceeded that of sericin. In the Student Test, there is a significant difference between NC and sericin, NC and RBP55 (0.1 g / L) at the 5% level, and there is also a significant difference between sericin and RBP55 (0.1 g / L). Differences were noted. That is, both sericin and RBP55 were effective for cell culture, but RBP55 was found to be more effective.

(2-2) Examination of the optimal concentration of RBP55 The concentration of RBP55 was 0.025, 0.05, 0.1 and 0.2 g / L, and no addition and sericin (1 g / L) were used as controls. The number of cells was counted on the day.
The results are shown in FIG. As is clear from FIG. 2, it was more effective than sericin at a concentration of 0.2 g / L or less. The optimum concentration was found to be 0.1 g / L.

(2-3) Comparison with BSA (Bovine Serum Albumin) It is known that when BSA is added to ASF104 medium at 1 g / L, cell proliferation is greatly improved. Therefore, a medium supplemented with 0.1 g / L of both RBP55 and BSA was prepared, and after culturing for 2 days after seeding, the number of cells was counted.
The results are shown in FIG. As can be seen from FIG. 3, by adding 0.1 g / L or 0.3 g / L of RBP55, an effect equivalent to that obtained by adding 1 g / L of BSA was observed. When both were compared at the same concentration, it was shown that RBP55 exerted an effect equivalent to or better than BSA in a small amount. On the other hand, at the optimum concentration of BSA of 1 g / L, RBP55 was excessive and it was shown to work for inhibition.

(2-4) Examination of the optimal concentration of RBP74 The concentration of RBP74 was 0.025, 0.05, 0.1 and 0.2 g / L, and no addition and sericin (1 g / L) were used as controls. The number of cells was counted on the day.
The results are shown in FIG. As is clear from FIG. 4, 0.1 g / L and 0.2 g / L were more effective than sericin. The optimum concentration was found to be 0.2 g / L. The optimal concentration of RBP74 was higher than the optimal concentration of RBP55, suggesting that some of the components in the rice bran extract that promote cell growth are not proteins.

(2-5) Examination of effect on production of monoclonal antibody Cell culture supernatant was collected, and its antibody concentration was measured by sandwich ELISA. A 96-well assay plate manufactured by Sumitomo Bakelite was first coated with anti-mouse IgG antibody and then blocked with 5% skim milk. After washing, a culture supernatant sample and a standard mouse IgG antibody diluted appropriately with a 1% skim milk solution were added and incubated. Thereafter, anti-mouse IgG antibody modified with peroxidase enzyme was added and incubated. After washing, orthophenylenediamine dihydrochloride was added together with hydrogen peroxide, and the antibody concentration was evaluated by the amount of coloration.
The results are shown in FIG. As is clear from FIG. 5, when RBP55 was added at a concentration of 0.3 g / L (300 μg / mL), the production amount of monoclonal antibody showed an increasing tendency as compared with the case where RBP55 was not added.

(2-6) Examination of cell growth promoting effect by rice bran extract subjected to autoclaving sterilization Using a medium supplemented with 0.2 g / L of RBP74 autoclaved (121 ° C., 15 minutes), 3 days after seeding The number of cells was counted. As controls, a medium supplemented with RBP74 and a medium supplemented with 0.2 g / L of RBP74 not autoclaved were used.
The results are shown in Table 1. As is clear from Table 1, the cell growth promoting activity of RBP74 was maintained even after high-pressure steam sterilization. This result revealed that the active ingredient having cell growth promotion is not a protein, and that the rice bran extract of Example 1 is highly useful as an additive that can be autoclaved after being added to the medium. .

(2-7) Examination of cell growth promoting effect by rice bran extract obtained by molecular weight fractionation RBP74 was fractionated based on molecular weight using an ultrafiltration membrane (Amicon Ultra, manufactured by Millipore). Using a 3 kDa membrane, a fraction of 3 kDa or less (hereinafter referred to as “3K or less”) and a fraction of 3 kDa or more (hereinafter referred to as “3K or more”) were obtained. Further, using a 10 kDa membrane, a fraction of 10 kDa or less (hereinafter referred to as “10K or less”) and a fraction of 10 kDa or more (hereinafter referred to as “10K or more”) were obtained.
Additive-free and unfractionated RBP74 served as controls. The concentration was 0.2 g / L of unfractionated RBP74, and each fraction sample was added to the medium so as to contain the amount contained in 0.2 g / L of unfractionated RBP74. The number of cells was counted 3 days after seeding.

  The results are shown in FIG. As is clear from FIG. 6, it was shown that 3K or less and 10K or more are effective, and the effect is higher than unfractionated.

(2-8) Examination of cell growth promotion effect by rice bran extract subjected to molecular weight fractionation and autoclave sterilization For autoclave sterilization of 3K or less and 10K or more that showed higher effect than unfractionated in (2-5) above It was examined whether the cell growth promoting effect was changed by the treatment. With no addition and unfractionated RBP74 as controls, 3K or less, high-pressure steam sterilization treatment 3K or less, 10K or more, high-pressure steam sterilization treatment 10K or more were added to the medium, and the number of cells was counted on the third day after seeding. Similar to (2-5), the concentration was 0.2 g / L of unfractionated RBP74, and each fractioned sample was added to the medium so as to contain the amount contained in 0.2 g / L of unfractionated RBP74. .

  The results are shown in FIG. As is clear from FIG. 7, the effect disappeared by high-pressure steam sterilization at 3K or less, but the effect was enhanced by high-pressure steam sterilization at 10K or more.

[Example 3: Examination of effects on CHO cells]
(3-1) Effect of RBP55 on cell proliferation CHO-DP12 (ATCC No. CRL-12445) was used for the cells, and ASF104 medium (Ajinomoto) was used for the medium. Cells were seeded at a concentration of 4 × 10 ⁴ cells / mL and cultured for several days according to a standard method, and then the number of cells was counted.

The concentration of RBP55 was 0 (control), 0.001, 0.03, 0.1, 0.3 and 1 g / L, and the number of cells was counted on the third day after seeding.
The results are shown in FIG. As is clear from FIG. 8, the growth was significantly promoted at the concentrations of 0.1 and 0.3 g / L as compared with the control group. On the other hand, cytotoxicity was observed at 1 g / L.

(3-2) Examination of effect on recombinant protein production Cell culture supernatant was collected, and its antibody concentration was measured by sandwich ELISA. A 96-well assay plate manufactured by Sumitomo Bakelite was first coated with anti-human IgG antibody and then blocked with 5% skim milk. After washing, a culture supernatant sample and a standard human IgG antibody diluted appropriately with a 1% skim milk solution were added and incubated. Thereafter, an anti-human IgG antibody modified with peroxidase enzyme was added and incubated. After washing, orthophenylenediamine dihydrochloride was added together with hydrogen peroxide, and the antibody concentration was evaluated by the amount of coloration.
The results are shown in FIG. As is apparent from FIG. 9, when RBP55 was added at a concentration of 0.1 g / L (100 μg / mL) or 0.3 g / L (300 μg / mL), the amount of recombinant protein produced was significantly higher than that without RBP55. Increased to.

[Example 4: Examination of effects on HepG2 cells]
HepG2 (ATCC No. HB-8065) was used for the cells, and ASF104 medium (Ajinomoto) was used for the medium. Cells were seeded at a concentration of 6 × 10 ⁴ cells / mL and cultured for several days according to a standard method, and then the number of cells was counted.

The concentration of RBP55 was 0 (control), 0.1, 0.3 and 1 g / L, and the number of cells was counted 3 days after seeding.
The results are shown in FIG. As is clear from FIG. 10, proliferation was significantly promoted at a concentration of 0.3 g / L compared to the control group.

[Example 5: Examination of effects on mesenchymal stem cells (MSC)]
A serum-free medium for mesenchymal stem cells called STK1 (Tucell, Inc.) is known. However, this medium loses its differentiation ability at an early stage as compared with the case where a medium containing serum is used. Therefore, it was investigated whether differentiation ability could be maintained by adding rice bran extract.

(1) Experimental Method MSCs were prepared from Wistar rats (3 weeks old, male) according to a conventional method. 300 cells / well were seeded in a 96-well plate under the following conditions. STK1 containing 100 μg / mL of RBP55 and 100 μL / well of STK1 containing no RBP55 were added. As positive controls, αMEM containing 15% fetal bovine serum containing 100 μg / mL of RBP55 and 100 μL / well of αMEM containing 15% fetal bovine serum not containing 100 μg / mL of RBP55 were added. In each group, n = 3. After subcultured twice under these culture conditions, a differentiation experiment was performed. In the positive control and the culture with STK1 not containing RBP55, the cells were adhered, and when the cell density became confluent, bone differentiation was induced. In the culture with STK1 containing RBP55, aggregates were formed, and bone differentiation was induced after the formation of aggregates. The differentiation induction period was 21 days, and the medium was changed every 3 days. Bone differentiation was evaluated by alizarin red staining.

(2) Results The results are shown in FIG. As is clear from FIG. 11, it was shown that the differentiation ability can be maintained by adding RBP55 to STK1.

  The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope shown in the claims, and technical means disclosed in different embodiments are appropriately combined. The obtained embodiment is also included in the technical scope of the present invention. Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.

Claims (7)

  A cell growth promoter comprising a rice bran extract as an active ingredient.   The cell growth promoter according to claim 1, wherein the rice bran extract contains 50% by mass or more of protein.   A culture medium additive comprising the cell growth promoting agent according to claim 1 or 2.   A cell culture medium comprising the cell growth promoter according to claim 1 or 2.   The medium according to claim 4, wherein the cell is an animal cell.   The medium according to claim 5, wherein the animal cell is a mammalian cell.   The medium according to claim 6, wherein the mammalian cell is a hybridoma, a recombinant protein-producing cell or a stem cell.