Cytostatics

Abstract

The present disclosure pertains to a cell growth regulator which contains glutamic acid, and a method for controlling cell growth which uses the same.

Description

REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims priority to Japanese Patent Application No. 2019-239572, filed on December 27, 2019, the entire disclosure of which is incorporated herein by reference. [Technical field]

[0002] The present disclosure relates to a cell proliferation inhibitor comprising glutamic acid and a method for controlling cell proliferation using the same. [Background technology]

[0003] In biology, cultured cells that are artificially cultivated outside the body are widely used in in vitro experimental systems, regenerative medicine, and cell therapy. In order to maintain the properties of the cells at a constant level, they need to be subcultured to continue to grow in the medium. However, for non-established cells that have a limited number of subcultures (life span), such as primary cultured cells and finite cell lines, the properties of the cells often change when they are subcultured.

[0004] Therefore, cryopreservation is the general method for maintaining the properties of cultured cells for a medium to long period of time. However, cryopreservation causes significant damage to cells that are sensitive to freezing and thawing, such as primate cells.

[0005] In addition, when using fast-growing cells or slow-growing cultured cells, it is difficult to adjust the cell concentration to the desired level, and skill is required. A method for suppressing cell proliferation by temporarily changing the environmental temperature of the cells to a low temperature such as 4°C is also known, but this can only be maintained for a few hours.

[0006] Furthermore, it is known that among cultured cells, stem cells lose their undifferentiated state (differentiation ability), which is one of the properties of the cells, through subculture. Therefore, in order to maintain the undifferentiated state, it was necessary to add, for example, LIF (leukemia inhibitory factor) to mouse ES cells. Also, in the case of primate ES cells, it was necessary to add an undifferentiated state maintenance factor or to culture the cells in the presence of feeder cells (see JP 2007-228815 A (Patent Document 1)). However, many of these undifferentiated state maintenance factors are expensive, and the co-culture with feeder cells was complicated.

[0007] Therefore, there is a demand for a method for easily controlling cell proliferation while maintaining the properties of the cells, without the need for subculturing the cells, other than by cryopreservation.

[0008] As a method other than cryopreservation, for example, JP 2008-104407 A (Patent Document 2) discloses a method for preserving adherent cells, characterized by preserving the cells under refrigerated conditions using a cell preservation solution containing reducing sugars. However, this method requires refrigeration at a low temperature, and the types of cells that can be preserved are limited.

[0009] Xylose is one of the sugars that make up glycans and plays an important role in living organisms, such as in intercellular communication. Xylose is also known to be abundant in woody biomass, and there is a demand for expanding its use in various fields as an unused resource.

[0010] The present inventors have previously confirmed the effects of various carbohydrates (glucose, xylose, galactose, etc.) on mouse ES cells and reported the possibility that xylose can maintain the undifferentiated state in mouse ES cells (Tadayuki Yokoyama et al., "Effects of mouse ES cell proliferation and EB formation in environments containing various sugars," Abstracts of the 7th General Meeting of the Japanese Society for Regenerative Medicine, Vol. 7, pp. 239, 2008 (Non-Patent Document 1); Tadayuki Yokoyama et al., "Investigation of the effectiveness of maintaining the undifferentiated state of mouse ES cells in a xylose-containing environment," Abstracts of the 8th General Meeting of the Japanese Society for Regenerative Medicine, Vol. 8, pp. 221, 2009 (Non-Patent Document 2)).

[0011] Furthermore, the present inventors have reported that when pluripotent stem cells are cultured in a medium in which the glucose contained in typical medium is replaced with xylose, the cells can maintain their survival and suppress cell proliferation for a long period of more than one week at normal culture temperatures, despite the fact that it was previously thought that the cells would not be able to survive because they cannot use sugars other than glucose as energy (Patent Document 3).

[0012] However, it remains an important challenge to provide a method for easily controlling the proliferation of a wide range of primate cells, including fibroblasts and mesenchymal stem cells, while maintaining their properties. [Prior art documents] [Patent documents]

[0013] [Patent Document 1] JP 2007-228815 A [Patent Document 2] JP 2008-104407 A [Patent Document 3] Patent No. 6042099 [Non-patent literature]

[0014] [Non-Patent Document 1] Tadayuki Yokoyama et al., "Effects of Various Sugar-Containing Environments on the Growth and EB Formation of Mouse ES Cells," Abstracts of the 7th Annual Meeting of the Japanese Society for Regenerative Medicine, Vol.7, pp.239, 2008 [Non-Patent Document 2] Tadayuki Yokoyama et al., "Effectiveness of maintaining undifferentiated state of mouse ES cells in a xylose-containing environment," Abstracts of the 8th Annual Meeting of the Japanese Society for Regenerative Medicine, Vol.8, pp.221, 2009 Summary of the Invention

[0015] The present inventors have now found that the use of a specific amino acid as a cell proliferation inhibitor makes it possible to easily control the proliferation of a wide range of primate cells, including fibroblasts and mesenchymal stem cells, while maintaining their properties. The present disclosure is based on this finding.

[0016] Therefore, an object of the present disclosure is to provide a cell proliferation inhibitor and a method for controlling cell proliferation using the same.

[0017] According to one embodiment of the present disclosure, the cell proliferation inhibitor comprises glutamic acid.

[0018] According to one aspect of the present disclosure, there is provided a method for controlling cell proliferation, comprising maintaining cells using a medium according to the present disclosure.

[0019] According to the cell proliferation inhibitor of the present disclosure, cell proliferation can be inhibited while maintaining the properties of the cells under normal culture conditions. In addition, the proliferation inhibitor of the present disclosure can be advantageously used to inhibit cell proliferation while inhibiting cell death. Therefore, the cell proliferation inhibitor of the present disclosure is extremely useful for short-term storage and maintenance of cells without requiring cryopreservation.

[0020] Furthermore, the method for controlling cell proliferation according to the present disclosure requires only the procedure of replacing an ordinary medium with a medium containing the cell proliferation inhibitor of the present disclosure, and therefore does not require skill and is extremely simple.

[0021] Furthermore, when the cells are pluripotent stem cells, the use of a medium containing the cell proliferation inhibitor of the present disclosure allows the cells to be maintained while suppressing their proliferation, and allows the cells to maintain their undifferentiated and totipotent state without the addition of factors for maintaining undifferentiation or without culturing in the presence of feeder cells. Therefore, even if the pluripotent stem cells are sensitive to freezing, they can be maintained for a short period of time while maintaining their undifferentiated and totipotent state, in the same way as if they were frozen. [Brief description of the drawings]

[0022] [Figure 1] The graph shows the results of microscopic observation of human fibroblasts cultured in cell maintenance media (xylose DMEM basal medium, glucose-free DMEM basal medium) with adjusted amounts of added glutamic acid (phase contrast image on the 9th day of culture, magnification 100x). [Diagram 2] The figures show the results of microscopic observation of human mesenchymal stem cells cultured in cell maintenance medium (xylose DMEM basal medium, glucose-free DMEM basal medium) with adjusted amounts of added glutamic acid (phase contrast image on the 9th day of culture, magnification 100x). [Diagram 3] The figures show the results of microscopic observation of human mesenchymal stem cells cultured in cell maintenance medium (xylose DMEM basal medium, glucose-free DMEM basal medium) conditioned with the addition of 8 mM glutamic acid (phase contrast image on the 12th day of culture, magnification 100x). Description of the Invention

[0023] Cytostatics As described above, the cell proliferation inhibitor according to the present disclosure comprises glutamic acid.

[0024] According to a preferred embodiment, the cell proliferation inhibitor according to the present disclosure contains glutamic acid as an active ingredient. In the present disclosure, the term "active ingredient" refers to an ingredient required to exert the cell proliferation inhibitory effect that is the object of the present disclosure. Here, the cell proliferation inhibitory effect means maintaining cell survival and inhibiting cell proliferation. By inhibiting cell proliferation, cells can be preserved.

[0025] The cytostatic agent according to the present disclosure is used in conjunction with xylose, as described above.

[0026] Xylose is a pentose monosaccharide that is abundant in woody biomass and is also called wood sugar. The xylose used in the present disclosure may be D-xylose, which is abundant in nature, or L-xylose or DL-xylose, which is prepared synthetically. The xylose used in the present disclosure is preferably D-xylose.

[0027] As used herein, the term "cells" includes fibroblasts, mesenchymal stem cells, pluripotent stem cells, cancer cells, established cells, primary cultured cells, and finite cell lines. The cells used in the present disclosure are preferably fibroblasts, mesenchymal stem cells, or pluripotent stem cells, more preferably fibroblasts or mesenchymal stem cells, in that they have a significantly high cell proliferation rate and must retain traits such as undifferentiated state.

[0028] Fibroblasts are one of the cells that make up connective tissue and include cells that produce components of the dermis such as collagen, elastin, and hyaluronic acid.

[0029] Mesenchymal stem cells are somatic stem cells derived from mesodermal tissue (mesenchyme). Mesenchymal stem cells are cells that can differentiate into cells belonging to the mesenchymal system, and are expected to be applied to regenerative medicine such as the reconstruction of bones, blood vessels, and cardiac muscle. Mesenchymal stem cells can be classified according to the tissue from which they are collected, and include bone marrow-derived stem cells and adipose tissue-derived stem cells.

[0030] Pluripotent stem cells include, for example, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), embryonic germ cells (EG cells), embryonic cancer cells (EC cells), and adult pluripotent stem cells (APS cells). Pluripotent stem cells may be derived from animals, insects, etc., but are preferably derived from mammals, and more preferably from primates. The pluripotent stem cells used in the present disclosure are preferably ES cells or iPS cells.

[0031] Here, "pluripotency" can also be said to mean multipotency, but it refers to the ability to differentiate into any differentiated cell belonging to the ectoderm, mesoderm, or endoderm, and the property of being able to differentiate into at least one differentiated cell belonging to each of the ectoderm, mesoderm, and endoderm, and may also include the ability to differentiate into germ cells. In addition, differentiated cells can differentiate into many types of cells that constitute tissues and organs in the body in the future. Whether or not a cell has pluripotency can be determined, for example, by co-culturing PA6 feeder cells and pluripotent cells and evaluating whether or not they are induced into neural cells, or by the expression of various differentiation markers.

[0032] "Undifferentiated" means that the stem cells have not yet differentiated into a specific cell lineage, and is a property of stem cells that are relatively high in the stem cell hierarchy. Whether or not the stem cells have undifferentiated properties can be determined by measuring undifferentiated cell markers such as Oct3 / 4, Nanog, and Rex-1.

[0033] The term "cancer cells" as used herein refers to cells derived from cancer. For example, cells derived from pancreatic adenocarcinoma, breast adenocarcinoma, and liver cancer are included. Specific examples include Panc1, Mia-Pa-Ca2, and MCF7.

[0034] The term "cell line" used in the present disclosure refers to an immortalized cell line. Examples include STO cells and SNL cells. By using the proliferation inhibitor of the present disclosure, unnecessary subculture can be eliminated.

[0035] The term "primary cultured cells" as used herein means cells that are initially seeded and cultured with tissues or cells collected from a living body. The term "finite cell line" as used herein means subcultured cells that have a limited number of subcultures (lifespan). By using the proliferation inhibitor of the present disclosure, primary cultured cells and finite cell lines, whose properties are easily changed, can be maintained and preserved.

[0036] The cell proliferation inhibitor of the present disclosure is expected to be used in substance response tests and clinical examinations in a state where vital activities are suspended.

[0037] According to one aspect of the present disclosure, there is provided a cell culture medium comprising the cell proliferation inhibitor of the present disclosure.

[0038] Here, the term "cell culture medium" refers to a culture medium or composition thereof suitable for culturing the above-mentioned cells, which allows a certain number of cells to survive for a certain period of time while maintaining the properties of the cells and without causing any significant damage to the survival of the cells. The cell culture medium may be in powder or liquid form.

[0039] The content of glutamic acid in the cell culture medium can be appropriately changed depending on the type of cells to be cultured, the purpose of the culture, the type of basal medium, etc. The content of glutamic acid in the basal medium can be, for example, 0.5 μM or more, and is preferably 0.5 μM to 10 mM, more preferably 0.5 mM to 10 mM, even more preferably 1 to 10 mM, even more preferably 5 mM to 8 mM, and even more preferably 8 mM.

[0040] Here, examples of the basal medium include Dulbecco's modified Eagle's medium (DMEM), Eagle's minimum essential medium (MEM), α-modified Eagle's minimum essential medium (α-MEM), Ham's F12 medium, MCDB medium, Fisher's medium, and RPMI-1640 medium, in which glucose in the medium is replaced with xylose, with DMEM being preferred.

[0041] The content of xylose in the cell culture medium is not particularly limited, and can be appropriately changed depending on the type of cells to be cultured, the purpose of the culture, the type of the basal medium, etc. The content of xylose in the medium may be the same as the amount of glucose in a normal basal medium, for example, 0.1 to 10.0 g / L, but is preferably 0.5 to 10.0 g / L, more preferably 0.5 to 5.0 g / L, even more preferably 0.8 to 5.0 g / L, and even more preferably 0.8 to 3.5 g / L. Even if the content of xylose in the medium according to the present disclosure is small, the present disclosure can show a sufficient effect, but since xylose is non-toxic and has excellent water solubility, there is usually no substantial problem even if it is added in a large amount.

[0042] In addition, from the viewpoint of suppressing cell growth due to glucose, it is preferable that the cell culture medium is used without adding glucose. Thus, according to one embodiment, the cell culture medium does not substantially contain glucose.

[0043] When xylose is introduced into the medium, it is preferable to introduce xylose by substituting xylose for glucose in a medium used for conventional cell culture.

[0044] The medium may contain serum, serum substitutes, or other components as necessary. Any known serum or serum substitutes may be used. For example, serum may include FBS, FCS, etc., and serum substitutes may include KSR, etc.

[0045] Other ingredients include non-essential amino acids, pH adjusters, etc.

[0046] The serum or serum substitute or other components added to the medium are preferably those from which carbohydrates have been removed by membrane treatment, for example, using a dialysis membrane 36 / 32 (manufactured by EIDIA Co., Ltd.).

[0047] The cell culture medium is preferably used as a cell maintenance medium. Here, the term "cell maintenance medium" refers to a culture medium or a composition thereof suitable for culturing the above-mentioned cells, which allows the cells to survive at a certain number for a certain period of time while maintaining the characteristics of undifferentiated cells, such as the undifferentiated state and pluripotency of undifferentiated cells and the collagen production ability of fibroblasts.

[0048] Since the cell maintenance medium of the present disclosure contains the cell proliferation inhibitor, it can maintain the properties of cells even if it does not substantially contain factors for maintaining undifferentiation other than glutamic acid and xylose, which are considered to be essential components of cell culture media. Therefore, according to one embodiment of the present disclosure, the medium of the present disclosure contains a cell proliferation inhibitor and does not substantially contain factors for maintaining undifferentiation other than glutamic acid and xylose. Examples of factors for maintaining undifferentiation other than glutamic acid and xylose include bFGF (basic fibroblast growth factor), LIF, and feeder cell-derived components.

[0049] Furthermore, according to one aspect of the present disclosure, there is provided a method for controlling cell proliferation, comprising maintaining cells using the cell maintenance medium of the present disclosure.

[0050] Here, "control of cell proliferation" means stopping the proliferation ability of cells at a desired time, maintaining the cell concentration without causing any serious damage to the cells that affects their survival, and then resuming cell proliferation at a desired time. Specifically, cell proliferation can be controlled by replacing the cell maintenance medium of the present disclosure with a normal cell culture medium. Here, depending on the cell, the proliferation speed after proliferation resumption can be made to be the same level as or higher than that of normal culture.

[0051] Cell growth control may be carried out at normal cell culture temperatures and environmental conditions.

[0052] The period for suppressing cell proliferation can be appropriately changed depending on the type of cells to be cultured, the purpose of the culture, the type of basal medium, the culture temperature, etc. For example, when the cell is cultured under normal culture conditions using DMEM in which glucose in the medium is replaced with xylose as the cell maintenance medium of the present disclosure, it is expected that cell proliferation can be suppressed for at least one month, and preferably for at least 26 days, more preferably for at least 21 days, even more preferably for at least 14 days, and even more preferably for at least 8 days. The cell maintenance medium may be exchanged during the growth inhibition period. By exchanging the medium, amino acids and protein components in the medium can be replenished, even during long-term culture, which has the advantage of maintaining the properties of the cells.

[0053] Furthermore, according to one embodiment of the present disclosure, the following (1) to (8) are provided. (1) A cell proliferation inhibitor comprising glutamic acid. (2) The cell proliferation inhibitor according to (1) for use together with xylose. (3) The cell proliferation inhibitor according to (1) or (2), wherein the cells are selected from fibroblasts, mesenchymal stem cells, and pluripotent stem cells. (4) The cell proliferation inhibitor according to any one of (1) to (3), wherein the cell is a human cell. (5) A cell maintenance medium containing 0.5 μM or more of glutamic acid. (6) The cell maintenance medium according to (5), further comprising xylose. (7) The cell maintenance medium according to (5) or (6), which is substantially free of factors for maintaining undifferentiated cells other than glutamic acid and xylose. (8) A method for controlling cell proliferation, comprising maintaining cells using the medium according to any one of (5) to (7). EXAMPLES

[0054] The present disclosure will now be described in detail with reference to examples. In addition, unless otherwise specified, the units and measurement methods in this disclosure follow the provisions of the Japanese Industrial Standards (JIS). In addition, unless otherwise specified, "parts" and "%" mean "parts by mass" and "% by mass", respectively, and temperatures mean Celsius.

[0055] Glutamic acid addition test (1) Preparation of medium The basal cell culture media were prepared to have the following compositions:

[0056] DMEM basal medium (hereinafter also referred to as "w / Glc") (Glucose content 1.0g / L, xylose content 0g / L) Dulbecco's modified Eagle's medium (DMEM; Cell Science Institute, Inc.) was adjusted in terms of the amount of each component added to give 10% by volume dialyzed FBS (GE Healthcare), 50 U / mL penicillin, and 50 μg / mL streptomycin (Fujifilm Wako Pure Chemical Industries, Ltd.).

[0057] Cell Maintenance Media (i) Xylose DMEM basal medium (hereinafter also referred to as "w / Xy") (Glucose content 0g / L, xylose content 1.0g / L) In a modified DMEM (M-DMEM, obtained from Cell Science Institute, Inc.) in which glucose in the DMEM basal medium was replaced with D-xylose, the amount of each component added was adjusted to 10% by volume dialyzed FBS, 50 U / mL penicillin, and 50 μg / mL streptomycin (Fujifilm Wako Pure Chemical Industries), to obtain w / Xy.

[0058] (ii) Glucose-free DMEM basal medium (hereinafter also referred to as "w / o Glc") (Glucose content 0g / L, xylose content 0g / L) A modified DMEM basal medium (M-DMEM, obtained from Cell Science Institute, Inc.) was used to remove glucose from the DMEM medium. The amount of each component added was adjusted to 10% by volume of dialyzed FBS, 50 U / mL penicillin, and 50 μg / mL streptomycin (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain w / o Glc.

[0059] (2)Cells Preparation of human fibroblast cells Human fibroblasts (catalog number CC-2511) were cultured in a gelatin-coated cell culture dish (100 mm culture dish, obtained from Sanplatec Co., Ltd.) at 37°C in the presence of 5% CO. DMEM basal medium was used as the medium.

[0060] Preparation of human mesenchymal stem cells Human mesenchymal stem cells (manufactured by Lonza, catalog number PT-5006) were cultured at 37°C in the presence of 5% CO2 in a cell culture dish (100 mm culture dish, obtained from Sanplatec Co., Ltd.) coated with 10 μg / ml Fibronectin (Promocell). DMEM basal medium was used.

[0061] (3) Cell proliferation inhibition test Example 1 After culturing the cells, the cells were harvested using a 0.1 wt% trypsin / 1 mM EDTA solution, seeded onto a 12-well plate (obtained from Corning Corporation), and cultured overnight at 37°C in the presence of 5% CO2 using the above-mentioned DMEM basal medium.

[0062] The DMEM basal medium was replaced with a desired cell maintenance medium, and the cells were cultured (referred to as "day 0 after culture"). The medium was not replaced during the culture period.

[0063] Furthermore, a culture test similar to that described above was carried out, except that 1 mM, 5 mM or 8 mM glutamic acid was added to the basal cell culture medium.

[0064] Observation of cell status On the 9th day after culturing, the cells were observed under a phase contrast microscope (IX71, Olympus, magnification 100x).

[0065] The results when human fibroblasts were used are shown in FIG. In the xylose DMEM basal medium (w / Xy), it was observed that as the concentration of glutamic acid increased, the number of dead cells decreased and cell proliferation was suppressed. In particular, almost no dead cells were observed when the glutamic acid concentration was in the range of 1 to 8 mM.

[0066] It was also observed that the number of dead cells decreased and cell proliferation was suppressed as the concentration of glutamic acid increased in the glucose-free DMEM basal medium (w / o Glc).

[0067] Furthermore, the results when human mesenchymal stem cells were used are shown in FIG. 2, and a similar tendency was observed as when human fibroblasts were used. In the xylose DMEM basal medium (w / Xy), it was observed that the number of dead cells decreased and cell proliferation was suppressed as the concentration of glutamic acid increased. In particular, almost no dead cells were observed when the glutamic acid concentration was in the range of 5 to 8 mM.

[0068] It was also observed that the number of dead cells decreased and cell proliferation was suppressed as the concentration of glutamic acid increased in the glucose-free DMEM basal medium (w / o Glc).

[0069] In w / Xy, the cells were in good condition and the number of dead cells was significantly less than that in w / o Glc.

[0070] As a control test, a cell proliferation inhibitory effect confirmation test was conducted using the same procedure as above, except that DMEM basal medium was used instead of the cell maintenance medium. When either human fibroblasts or human mesenchymal stem cells were used, almost no cell death was observed, but cell proliferation was confirmed.

[0071] Example 2 The same culture test as in Example 1 was carried out, except that the medium was changed every 3 days during the culture period.

[0072] The cell numbers for each day of culture are shown in Tables 1 and 2 below. Table 1 shows the results when human fibroblasts were used, and Table 2 shows the results when human mesenchymal stem cells were used. The cell numbers are the average values ​​of the cell counts measured in three wells by the following method. After culturing, the culture supernatant of the cells was removed by aspiration, and after washing with PBS, the cells were detached using a 0.1% trypsin / 1 mM EDTA solution (Fujifilm Wako Pure Chemical Industries, Ltd.). The reaction of the detachment solution was stopped by adding medium, and the cells were collected and counted. TM Cell counting was performed using a Thermo Fisher Scientific II Automated Cell Counter.

[0073] [Table 1]

[0074] As can be seen from Table 1, in human fibroblasts, the addition of glutamic acid was able to suppress cell proliferation up to 6 days after the control test using w / Glc medium. Furthermore, when w / Xy medium was used instead of w / Glc medium, the inhibitory effect on cell proliferation was more pronounced, and cell proliferation was suppressed for a longer period of time. On the other hand, when w / o Glc medium was used instead of w / Glc medium, the number of cells was significantly lower after 12 days, unlike w / Xy medium, and cell death occurred.

[0075] [Table 2]

[0076] From Table 2, it was confirmed that in human mesenchymal stem cells, the addition of glutamic acid was able to suppress cell proliferation up to 18 days after the control test using w / Glc medium. Furthermore, as with human fibroblasts, the effect of suppressing cell proliferation was significant when w / Xy medium was used instead of w / Glc medium. Furthermore, when w / o Glc medium was used instead of w / Glc medium, as shown in Figure 3, unlike w / Xy medium, the proportion of cells that became white around the cells was high, and the condition of the cells deteriorated.

[0077] The cell proliferation inhibitor of the present disclosure, which comprises glutamic acid, can inhibit cell proliferation. When used in combination with xylose, the cell proliferation inhibitor of the present disclosure can significantly inhibit cell proliferation and can inhibit cell proliferation for a longer period of time. Therefore, the cell proliferation inhibitor according to the present disclosure can be said to be extremely useful for short-term storage and maintenance of cells without the need for cryopreservation.

Claims

1. A cell proliferation inhibitor for cells selected from human fibroblasts and human mesenchymal stem cells, comprising glutamic acid and xylose.

2. A cell maintenance medium for cells selected from human fibroblasts and human mesenchymal stem cells, comprising glutamic acid and xylose.

3. The cell maintenance medium according to claim 2 , which is substantially free of factors for maintaining undifferentiated cells other than glutamic acid and xylose.

4. A method for controlling cell proliferation, comprising maintaining cells using the medium according to claim 2 or 3.

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