JP4624033B2 - Method for culturing mesenchymal stem cells and method for using hepatocyte growth factor - Google Patents
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この発明は、間葉系幹細胞の培養方法および肝細胞増殖因子の使用方法に関するものである。 The present invention relates to a method for culturing mesenchymal stem cells and a method for using hepatocyte growth factor.
骨髄液等に含まれている間葉系幹細胞は、骨、軟骨、脂肪等に分化可能な多分化能を有しているため、細胞治療や再生医療の細胞ソースとして注目を集めている。しかしながら、骨髄液等に含まれている間葉系幹細胞はごく微量であるため、臨床治療に用いる場合には、骨髄液内から集めた間葉系幹細胞を短期間で大量に増殖させることが重要である。 Mesenchymal stem cells contained in bone marrow fluid and the like are attracting attention as a cell source for cell therapy and regenerative medicine because they have multipotency capable of differentiating into bone, cartilage, fat and the like. However, since the amount of mesenchymal stem cells contained in bone marrow fluid is very small, it is important to proliferate a large amount of mesenchymal stem cells collected from the bone marrow fluid in a short period of time when used for clinical treatment. It is.
従来、間葉系幹細胞を効率よく増殖させる方法として、例えば、特許文献１および特許文献２に開示されている方法が知られている。
これらの方法は、間葉系幹細胞の増殖能刺激物質として繊維芽細胞増殖因子を培地に添加するものである。
In these methods, fibroblast growth factor is added to a culture medium as a mesenchymal stem cell growth ability stimulating substance.
これに対して、本発明者は、試験、研究の結果、間葉系幹細胞をさらに短期間で大量に増殖させる増殖因子を見いだした。
本発明は上述した事情に鑑みてなされたものであって、患者から採取した間葉系幹細胞を短期間で大量に増殖させることができ、大量の間葉系幹細胞を必要とする臨床治療に貢献し得る間葉系幹細胞の培養方法および肝細胞増殖因子の使用方法を提供することを目的としている。
On the other hand, as a result of tests and studies, the present inventor has found a growth factor that allows mesenchymal stem cells to proliferate in large quantities in a shorter period of time.
The present invention has been made in view of the circumstances described above, and can proliferate a large amount of mesenchymal stem cells collected from a patient in a short period of time, contributing to clinical treatment requiring a large amount of mesenchymal stem cells. An object of the present invention is to provide a method for culturing mesenchymal stem cells and a method for using hepatocyte growth factor.
上記目的を達成するために、本発明は、以下の手段を提供する。
本発明の参考例は、肝細胞増殖因子を１０ｎｇ／ｍｌ〜１００ｎｇ／ｍｌの濃度で添加した培地を提供する。
上記発明においては、肝細胞増殖因子を２０ｎｇ／ｍｌの濃度で添加することが好ましい。
In order to achieve the above object, the present invention provides the following means.
The reference example of the present invention provides a medium supplemented with hepatocyte growth factor at a concentration of 10 ng / ml to 100 ng / ml.
In the above invention, it is preferable to add hepatocyte growth factor at a concentration of 20 ng / ml.
また、上記発明においては、ビタミンＣを２５μｇ／ｍｌ〜４０００μｇ／ｍｌの濃度で添加することが好ましい。
ビタミンＣを添加した場合には、肝細胞増殖因子を４０ｎｇ／ｍｌの濃度で添加することが好ましい。
Moreover, in the said invention, it is preferable to add vitamin C by the density | concentration of 25 microgram / ml-4000 microgram / ml.
When vitamin C is added, it is preferable to add hepatocyte growth factor at a concentration of 40 ng / ml.
本発明は、肝細胞増殖因子及びビタミンＣを間葉系幹細胞の増殖刺激物質として添加した培地において間葉系幹細胞を培養する間葉系幹細胞の培養方法を提供する。
この方法においては、前記培地内における肝細胞増殖因子の濃度が、１０ｎｇ／ｍｌ〜１００ｎｇ／ｍｌであることが好ましい。
また、前記培地内における肝細胞増殖因子の濃度が２０ｎｇ／ｍｌであることがさらに好ましい。
The present invention provides a method for culturing mesenchymal stem cells in which mesenchymal stem cells are cultured in a medium to which hepatocyte growth factor and vitamin C are added as growth stimulating substances for mesenchymal stem cells .
In this method, the concentration of hepatocyte growth factor in the medium is preferably 10 ng / ml to 100 ng / ml.
More preferably, the concentration of hepatocyte growth factor in the medium is 20 ng / ml.
また、この場合には、前記培地内におけるビタミンＣの濃度が２５μｇ／ｍｌ〜４０００μｇ／ｍｌであることが好ましい。
また、ビタミンＣを添加した場合には、前記培地内における肝細胞増殖因子の濃度が４０ｎｇ／ｍｌであることが好ましい。
In this case, the concentration of vitamin C in the medium is preferably 25 μg / ml to 4000 μg / ml.
When vitamin C is added, the concentration of hepatocyte growth factor in the medium is preferably 40 ng / ml.
さらに、本発明は、培地内に骨髄液を投入して培養することにしてもよい。骨髄液内に含まれる造血幹細胞の増殖が、間葉系幹細胞と同様に肝細胞増殖因子の作用によって促進される。造血幹細胞は間葉系幹細胞の増殖を促進することが知られているので、本発明によれば、患者から採取した骨髄液の初代培養においても効率的に間葉系幹細胞を増殖させることができる。 Further, in the present invention, the bone marrow fluid may be introduced into the medium and cultured. The proliferation of hematopoietic stem cells contained in the bone marrow fluid is promoted by the action of hepatocyte growth factor as in the case of mesenchymal stem cells. Since hematopoietic stem cells are known to promote proliferation of mesenchymal stem cells, according to the present invention, it is possible to efficiently proliferate mesenchymal stem cells even in primary culture of bone marrow fluid collected from patients. .
また、本発明は、間葉系幹細胞の増殖能刺激物質として肝細胞増殖因子及びビタミンＣを使用する肝細胞増殖因子の使用方法を提供する。肝細胞増殖因子及びビタミンＣによって間葉系幹細胞の増殖を促進することができる。 The present invention also provides a method for using a hepatocyte growth factor using hepatocyte growth factor and vitamin C as a mesenchymal stem cell proliferation stimulating substance. Growth of mesenchymal stem cells can be promoted by hepatocyte growth factor and vitamin C.
本発明によれば、間葉系幹細胞を効率的に増殖して、患者から採取した骨髄液等に含まれる微量の間葉系幹細胞から、短期に、かつ大量に間葉系幹細胞を得ることができるという効果を奏する。 According to the present invention, it is possible to efficiently proliferate mesenchymal stem cells and obtain mesenchymal stem cells in a short time and in large quantities from a trace amount of mesenchymal stem cells contained in bone marrow fluid collected from a patient. There is an effect that can be done.
以下、本発明の一参考実施形態に係る培地、間葉系幹細胞の培養方法および肝細胞増殖因子の使用方法について説明する。
本実施形態に係る培地は、ＤＭＥＭ（Dulbecco's Modified Eagle Medium）に１０％のＦＢＳ（Fetal Bovine Serum）またはヒト血清、１０〜１００ｎｇ／ｍｌの肝細胞増殖因子（Hepatocyte Growth Factor：以下、単にＨＧＦという。）および抗生物質を混合したものである。
Hereinafter, a culture medium, a method for culturing mesenchymal stem cells, and a method for using hepatocyte growth factor according to one reference embodiment of the present invention will be described.
The medium according to this embodiment is DMEM (Dulbecco's Modified Eagle Medium), 10% FBS (Fetal Bovine Serum) or human serum, and 10 to 100 ng / ml hepatocyte growth factor (hereinafter simply referred to as HGF). ) And antibiotics.
本実施形態に係る間葉系幹細胞の培養方法は、培地として上記培地を使用して所定の培養条件（例えば、温度３７℃、湿度１００％、ＣＯ２濃度５％）で間葉系幹細胞の培養を行う方法である。
また、本実施形態に係るＨＧＦの使用方法は、このような培養方法において、ＨＧＦを間葉系幹細胞の増殖能刺激物質として使用する方法である。
本実施形態に係る培地、間葉系幹細胞の培養方法および肝細胞増殖因子の使用方法によれば、ＨＧＦの作用により、ＨＧＦを含まない場合と比較して間葉系幹細胞を大幅に増殖させることができるという効果がある。
The method for culturing mesenchymal stem cells according to this embodiment uses the above-mentioned medium as a medium and cultures mesenchymal stem cells under predetermined culture conditions (for example, temperature 37 ° C.,
In addition, the method of using HGF according to the present embodiment is a method of using HGF as a substance for stimulating the proliferation of mesenchymal stem cells in such a culture method.
According to the culture medium, the mesenchymal stem cell culturing method and the method of using hepatocyte growth factor according to the present embodiment, the mesenchymal stem cells are proliferated significantly by the action of HGF as compared with the case where HGF is not included. There is an effect that can be.
次に、本発明の一実施形態に係る培地、間葉系幹細胞の培養方法およびＨＧＦの使用方法について説明する。
本実施形態に係る培地は、ＤＭＥＭに１０％のＦＢＳまたはヒト血清、２５〜４０００μｇ／ｍｌのビタミンＣ、１０〜１００ｎｇ／ｍｌのＨＧＦおよび抗生物質を混合したものである。
Next, a culture medium, a method for culturing mesenchymal stem cells, and a method for using HGF according to an embodiment of the present invention will be described.
The medium according to the present embodiment is a mixture of DMEM with 10% FBS or human serum, 25 to 4000 μg / ml vitamin C, 10 to 100 ng / ml HGF, and antibiotics.
本実施形態に係る間葉系幹細胞の培養方法は、培地として上記培地を使用して所定の培養条件（例えば、温度３７℃、湿度１００％、ＣＯ２濃度５％）で間葉系幹細胞の培養を行う方法である。
また、本実施形態に係るＨＧＦの使用方法は、このような培養方法において、ＨＧＦを間葉系幹細胞の増殖能刺激物質として使用する方法である。
本実施形態に係る培地、間葉系幹細胞の培養方法および肝細胞増殖因子の使用方法によれば、ＨＧＦおよびビタミンＣの作用により、ビタミンＣを含まない場合と比較して間葉系幹細胞を大幅に増殖させることができるという効果がある。
The method for culturing mesenchymal stem cells according to this embodiment uses the above-mentioned medium as a medium and cultures mesenchymal stem cells under predetermined culture conditions (for example, temperature 37 ° C.,
In addition, the method of using HGF according to the present embodiment is a method of using HGF as a substance for stimulating the proliferation of mesenchymal stem cells in such a culture method.
According to the culture medium, the mesenchymal stem cell culturing method, and the method of using hepatocyte growth factor according to the present embodiment, the mesenchymal stem cells are greatly increased by the action of HGF and vitamin C as compared with the case of not containing vitamin C. There is an effect that can be proliferated.
［第１の参考実施例］
一参考実施形態に係る培地、間葉系幹細胞の培養方法およびＨＧＦの使用方法の第１の参考実施例を以下に説明する。
本実施例において使用された培地は、ＤＭＥＭに１０％のＦＢＳ、２０ｎｇ／ｍｌのＨＧＦおよび抗生物質を混合したものである。
[First Reference Example]
A first reference example of the culture medium, the mesenchymal stem cell culture method, and the method of using HGF according to one reference embodiment will be described below.
The medium used in this example is a mixture of 10% FBS, 20 ng / ml HGF and antibiotics in DMEM.
本実施例においては、まず、対数増殖中の間葉系幹細胞をトリプシンで回収し、細胞数１０６個／ｍｌとなるように１０％濃度のＦＢＳ入り培地で懸濁した。次いで、その細胞を３０００個／ｃｍ２となるように、６ウェルプレートに播いた。そして、上記のようにＤＭＥＭに１０％のＦＢＳ、２０ｎｇ／ｍｌのＨＧＦおよび抗生物質を混合した培地３ｍｌを加えた。 In this example, first, logarithmically growing mesenchymal stem cells were collected with trypsin and suspended in a medium containing FBS at a concentration of 10% so that the number of cells was 10 6 cells / ml. Next, the cells were seeded in a 6-well plate at 3000 cells / cm 2 . Then, 3 ml of medium in which 10% FBS, 20 ng / ml HGF and antibiotics were mixed with DMEM as described above was added.
このように調製された間葉系幹細胞をＣＯ ２ インキュベータ内に投入し、温度３７℃、湿度１００％、ＣＯ２濃度５％の培養条件下で１週間培養した。培養期間中、培地の交換を２回行った。培養３日目と７日目において細胞をトリプシンで回収して血球計算盤で細胞数を計測した。その結果を図１に示す。コントロールとして、ＨＧＦを含まない培地で同様の培養を行った結果を示す。 The mesenchymal stem cells thus prepared were put into a CO 2 incubator and cultured for 1 week under the culture conditions of a temperature of 37 ° C., a humidity of 100%, and a CO 2 concentration of 5%. During the culture period, the medium was changed twice. On the 3rd and 7th days of culture, the cells were collected with trypsin and the number of cells was counted with a hemocytometer. The result is shown in FIG. As a control, the results of the same culture in a medium not containing HGF are shown.
この実施例によれば、培地に２０ｎｇ／ｍｌのＨＧＦを添加した場合の方が、添加しないコントロールの場合と比較して、培養３日目および７日目のいずれの場合も、約１．５倍も多く間葉系幹細胞を増殖させることができた。 According to this example, the case where 20 ng / ml HGF was added to the medium was about 1.5 times in both cases on the third and seventh days of culture, compared to the case where the control was not added. Many times more mesenchymal stem cells could be proliferated.
［第２の参考実施例］
次に、一参考実施形態に係る培地、間葉系幹細胞の培養方法およびＨＧＦの使用方法の第２の参考実施例を以下に説明する。この実施例は、ＨＧＦの濃度を変化させて培養したものである。
[Second Reference Example]
Next, a second reference example of the medium, the method for culturing mesenchymal stem cells, and the method for using HGF according to one reference embodiment will be described below. In this example, the culture was performed while changing the concentration of HGF.
本実施例においては、まず、対数増殖中の間葉系幹細胞をトリプシンで回収し、細胞数１０６個／ｍｌとなるように１０％濃度のＦＢＳ入り培地で懸濁した。次いで、その細胞を３０００個／ｃｍ２となるように、１２ウェルプレートに播いた。そして、上記のようにＤＭＥＭに１０％のＦＢＳ、抗生物質およびそれぞれ０，１０，２０，４０，６０，１００ｎｇ／ｍｌのＨＧＦを混合した培地２ｍｌを加えた。 In this example, first, logarithmically growing mesenchymal stem cells were collected with trypsin and suspended in a medium containing 10% FBS so that the number of cells was 10 6 cells / ml. Next, the cells were seeded in a 12-well plate at 3000 cells / cm 2 . Then, 2 ml of medium mixed with 10% FBS, antibiotics and 0, 10, 20, 40, 60, and 100 ng / ml HGF, respectively, was added to DMEM as described above.
このように調製された間葉系幹細胞をＣＯ ２ インキュベータ内に投入し、温度３７℃、湿度１００％、ＣＯ２濃度５％の培養条件下で培養し、培養４日目に、トリプシンで回収した間葉系幹細胞の細胞数を血球計算盤によって計測した。その結果を図２に示す。
この実施例によれば、ＨＧＦが間葉系幹細胞の増殖を促進していること、および、その増殖促進効果がＨＧＦの濃度に依存してことがわかった。
The mesenchymal stem cells thus prepared were put into a CO 2 incubator, cultured under the culture conditions of a temperature of 37 ° C., a humidity of 100%, and a CO 2 concentration of 5%, and collected with trypsin on the 4th day of culture. The number of mesenchymal stem cells was counted with a hemocytometer. The result is shown in FIG.
According to this example, it was found that HGF promotes the proliferation of mesenchymal stem cells, and that the proliferation promoting effect depends on the concentration of HGF.
図２によれば、ＨＧＦ濃度が１０ｎｇ／ｍｌであっても、間葉系幹細胞の増殖促進効果が見られる。また、ＨＧＦ濃度が２０ｎｇ／ｍｌ以上となると、間葉系幹細胞の増殖促進効果はほぼ一定となっている。ＨＧＦ濃度があまりに高い場合には、間葉系幹細胞の健全性が害される可能性があり、現実的には１００ｎｇ／ｍｌ以下であることが好ましい。
したがって、間葉系幹細胞の増殖促進効果を得るためのＨＧＦ濃度としては、１０〜１００ｎｇ／ｍｌが好ましく、２０ｎｇ／ｍｌであることがさらに好ましい。
According to FIG. 2, even if the HGF concentration is 10 ng / ml, the mesenchymal stem cell proliferation promoting effect is observed. Further, when the HGF concentration is 20 ng / ml or more, the effect of promoting the proliferation of mesenchymal stem cells is almost constant. When the HGF concentration is too high, the soundness of the mesenchymal stem cells may be impaired, and in reality, it is preferably 100 ng / ml or less.
Therefore, the HGF concentration for obtaining the mesenchymal stem cell proliferation promoting effect is preferably 10 to 100 ng / ml, and more preferably 20 ng / ml.
［第１の実施例］
一実施形態に係る培地、間葉系幹細胞の培養方法およびＨＧＦの使用方法の第１の実施例を以下に説明する。この実施例は、ＨＧＦの濃度を変化させて培養したものである。
First Embodiment
A first example of a culture medium, a method for culturing mesenchymal stem cells, and a method for using HGF according to one embodiment will be described below. In this example, the culture was performed while changing the concentration of HGF.
本実施例においては、まず、対数増殖中の間葉系幹細胞をトリプシンで回収し、細胞数１０６個／ｍｌとなるように１０％濃度のＦＢＳ入り培地で懸濁した。次いで、その細胞を３０００個／ｃｍ２となるように、１２ウェルプレートに播いた。そして、上記のようにＤＭＥＭに１０％のＦＢＳ、抗生物質、５０μｇ／ｍｌのビタミンＣおよびそれぞれ０，１０，２０，４０，６０，１００ｎｇ／ｍｌのＨＧＦを混合した培地２ｍｌを加えた。 In this example, first, logarithmically growing mesenchymal stem cells were collected with trypsin and suspended in a medium containing 10% FBS so that the number of cells was 10 6 cells / ml. Next, the cells were seeded in a 12-well plate at 3000 cells / cm 2 . Then, 2 ml of medium in which 10% FBS, antibiotics, 50 μg / ml vitamin C and 0, 10, 20, 40, 60, and 100 ng / ml HGF were mixed with DMEM as described above was added.
このように調製された間葉系幹細胞をＣＯ ２ インキュベータ内に投入し、温度３７℃、湿度１００％、ＣＯ２濃度５％の培養条件下で培養し、培養４日目に、トリプシンで回収した間葉系幹細胞の細胞数を血球計算盤によって計測した。その結果を図３に示す。
図３は、図２に示した第２の実施例の結果を併せて示している。
図３によれば、間葉系幹細胞の培養において、ＨＧＦに加えてビタミンＣを併用することで、ビタミンＣを併用しない場合と比較して、間葉系幹細胞の増殖促進効果が向上していることがわかる。
The mesenchymal stem cells thus prepared were put into a CO 2 incubator, cultured under the culture conditions of a temperature of 37 ° C., a humidity of 100%, and a CO 2 concentration of 5%, and collected with trypsin on the 4th day of culture. The number of mesenchymal stem cells was counted with a hemocytometer. The result is shown in FIG.
FIG. 3 also shows the results of the second embodiment shown in FIG.
According to FIG. 3, in the culture of mesenchymal stem cells, the use of vitamin C in addition to HGF improves the effect of promoting the proliferation of mesenchymal stem cells compared to the case where vitamin C is not used in combination. I understand that.
また、ビタミンＣを添加した場合には、ＨＧＦが４０ｎｇ／ｍｌ以上の濃度において間葉系幹細胞の増殖促進効果が一定値となっている。したがって、ビタミンＣを添加した場合には、ＨＧＦの濃度を４０ｎｇ／ｍｌ以上とすることが好ましく、４０ｎｇ／ｍｌとすることがさらに好ましい。 In addition, when vitamin C is added, the growth promoting effect of mesenchymal stem cells is constant at a concentration of HGF of 40 ng / ml or higher. Therefore, when vitamin C is added, the concentration of HGF is preferably 40 ng / ml or more, and more preferably 40 ng / ml .
なお、ビタミンＣの濃度については、５０μｇ／ｍｌとした場合について実施したが、これに代えて、２５〜４０００μｇ／ｍｌの範囲において任意の濃度値を選択することにしてもよい。
また、上記各実施形態においては、対数増殖中の間葉系幹細胞について、ＨＧＦによる増殖促進効果を示したが、これに代えて、患者の体内から採取した骨髄液に対してＨＧＦおよび／またはビタミンＣを添加した培地を用いて、骨髄液内の間葉系幹細胞を培養することにしてもよい。この場合には、ＨＧＦによって骨髄液内に含有される造血幹細胞の増殖が促進され、これにより間葉系幹細胞の増殖が促進されることになる。このようにすることで、患者から採取した骨髄液の初代培養においても効率的に間葉系幹細胞を増殖させることができる。
In addition, about the density | concentration of vitamin C, although implemented about the case where it was set to 50 microgram / ml, you may decide to select arbitrary density | concentration values in the range of 25-4000 microgram / ml instead.
In each of the above embodiments, the growth promoting effect by HGF was shown for logarithmically growing mesenchymal stem cells. Instead, HGF and / or vitamin C was added to bone marrow fluid collected from the patient's body. The mesenchymal stem cells in the bone marrow fluid may be cultured using the added medium. In this case, the proliferation of hematopoietic stem cells contained in the bone marrow fluid is promoted by HGF, thereby promoting the proliferation of mesenchymal stem cells. By doing so, mesenchymal stem cells can be efficiently proliferated even in primary culture of bone marrow fluid collected from a patient.
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