JP2007167063A - Culture carrier for undifferentiated cell, culturing method and undifferentiated cultured cell - Google Patents
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/16—Particles; Beads; Granular material; Encapsulation
Abstract
Description
本発明は、ES細胞や成体多能性幹細胞等の多能性を有する未分化な細胞の培養技術、特に、培養担体およびこれを用いた培養方法ならびに未分化な3次元細胞塊を形成する培養細胞に関する。 The present invention relates to a technique for culturing pluripotent undifferentiated cells such as ES cells and adult pluripotent stem cells, in particular, a culture carrier, a culturing method using the same, and culturing to form undifferentiated three-dimensional cell clusters. Regarding cells.
疾患、老化、事故等による組織の損傷は、人間の生命活動にとって重大な影響を及ぼす。人間の組織の再生能力は低く、例えば、骨や軟骨等の修復は、人工骨等のインプラントによって損傷部を補充することにより行われている。 Tissue damage due to diseases, aging, accidents, etc. has a significant impact on human life activities. Human tissue has a low regenerative capacity. For example, bone and cartilage are repaired by replenishing the damaged part with an implant such as an artificial bone.
一方、近年の細胞培養技術においては、ヒト骨髄液からの未分化細胞の分離や、目的とする組織細胞への分化・誘導、3次元培養技術、足場材料の開発等の進歩により、体性幹細胞から皮膚、骨、軟骨、血管、心臓弁、靭帯等の組織を作製することが可能となり、一部は、既に臨床応用が始められている。 On the other hand, in recent cell culture technologies, somatic stem cells have been developed by the advancement of separation of undifferentiated cells from human bone marrow fluid, differentiation / induction to target tissue cells, three-dimensional culture technology, development of scaffold materials, etc. It is possible to produce tissues such as skin, bone, cartilage, blood vessels, heart valves, ligaments, etc., and some clinical applications have already begun.
このように、細胞培養によって、未分化な細胞から複雑な組織を得るためには、3次元構造を有する培養担体を用いて、ES細胞に代表される未分化細胞を、未分化な状態を保持したまま培養し、3次元細胞塊(コロニー)を形成させ、そして、この3次元細胞塊に、細胞増殖因子や栄養分を供給し、目的とする組織に分化・誘導させる必要がある。 Thus, in order to obtain complex tissues from undifferentiated cells by cell culture, undifferentiated cells represented by ES cells are maintained in an undifferentiated state using a culture carrier having a three-dimensional structure. It is necessary to culture as it is, to form a three-dimensional cell mass (colony), and to supply cell growth factors and nutrients to the three-dimensional cell mass to differentiate and induce the target tissue.
近年、このような未分化細胞から組織への分化・誘導の研究は、盛んに行われており、その一例として、生体安定性、生体親和性を有する気孔率75%のハイドロキシアパタイト多孔体内で未分化細胞を培養、増殖させた後、該多孔体を生体内に埋入することによって、細胞の働きを一層活発化させることが報告されている(例えば、特許文献1参照)。 In recent years, research on differentiation / induction from undifferentiated cells into tissues has been actively conducted, and as an example, it has not been performed in a hydroxyapatite porous body having a porosity of 75% having biostability and biocompatibility. It has been reported that after the differentiated cells are cultured and expanded, the porous body is embedded in the living body to further activate the cell function (see, for example, Patent Document 1).
また、ES細胞等に代表される未分化な細胞を培養、維持するために、培養担体として、プラスチックシャーレ上に、コラーゲン、ゼラチン、ラミニン、マトリゲル等の生体内成分をコーティングしたものが用いられたり、また、STO(マウス胎児繊維芽細胞)等に代表されるフィーダ細胞、もしくは、これに代わるES細胞支持細胞を培養して細胞層を形成させ、その上にES細胞を撒種(導入)し、培養する方法が行われている。
上記特許文献1に記載されているようなハイドロキシアパタイト等のリン酸カルシウム系セラミックスは、生体安定性、生体親和性に優れていることから、免疫拒絶反応を起こさない担体として好ましいと考えられる。
しかしながら、ハイドロキシアパタイトの主成分であるCa2+イオンやPO4 2-イオンは、骨芽細胞が骨形成を行う際の重要な因子であり、このため、ハイドロキシアパタイト等のリン酸カルシウム系セラミックスを、ES細胞等の未分化な細胞の培養担体として用いた場合、Ca2+イオン等の影響により、他の細胞に分化・誘導され、未分化なまま維持することが難しい。
Calcium phosphate ceramics such as hydroxyapatite described in Patent Document 1 are considered to be preferable as carriers that do not cause immune rejection because they are excellent in biostability and biocompatibility.
However, Ca 2+ ions and PO 4 2- ions, which are the main components of hydroxyapatite, are important factors when osteoblasts form bone. For this reason, calcium phosphate ceramics such as hydroxyapatite are used as ES. When used as a culture carrier for undifferentiated cells such as cells, it is difficult to maintain undifferentiated because it is differentiated and induced by other cells due to the influence of Ca 2+ ions and the like.
また、上述したようなフィーダ細胞等の支持細胞を用いる方法では、培養後、ES細胞と他の細胞を完全に分離できない可能性があり、しかも、生体内成分や支持細胞は、再生医療用の担体として用いた場合、危険因子の混入や免疫拒絶反応等のおそれがあり、臨床的に影響を及ぼすことが懸念される。
このため、フィーダ細胞等の支持細胞を使用することなく、培養担体上で、ES細胞を培養させる研究も進められてはいるが、未だ完全な3次元培養塊を形成できるまでには至っていない。
Further, in the method using feeder cells such as feeder cells as described above, there is a possibility that ES cells and other cells cannot be completely separated after culturing, and in vivo components and feeder cells are used for regenerative medicine. When used as a carrier, there is a risk of contamination with risk factors, immune rejection, and the like, and there is concern that it may have clinical effects.
For this reason, research for culturing ES cells on a culture carrier without using feeder cells such as feeder cells has been advanced, but a complete three-dimensional culture mass has not yet been formed.
したがって、ヒトのES細胞等を効率よく増殖させることができ、かつ、危険因子や免疫拒絶反応を生じることのない、未分化な3次元細胞塊を培養できる培養担体の開発が求められている。 Accordingly, there is a need for the development of a culture carrier that can efficiently proliferate human ES cells and the like and that can culture undifferentiated three-dimensional cell clusters that do not cause risk factors and immune rejection.
本発明は、上記技術的課題を解決するためになされたものであり、未分化な細胞を、免疫拒絶反応を起こさず、かつ、効率よく培養し、3次元細胞塊を形成することができる未分化細胞の培養担体および培養方法ならびに未分化なまま培養された3次元細胞塊を提供することを目的とするものである。 The present invention has been made in order to solve the above technical problem, and it is not yet possible to culture undifferentiated cells efficiently without causing immune rejection and to form a three-dimensional cell mass. It is an object of the present invention to provide a culture carrier and culture method for differentiated cells and a three-dimensional cell mass cultured undifferentiated.
本発明に係る未分化細胞の培養担体は、少なくとも細胞を撒種する部位が、孔径0.1μm以上10μm以下の微小孔を有するセラミックスであることを特徴とする。
上記のような微小孔は、セラミックスの粒子間に形成されるもので、細胞を撒種する部位に存在することにより、未分化細胞が培養担体に接着しやすく、また、培地の循環の促進も図られ、未分化細胞が3次元細胞塊状に形成されやすくなる。
The culture carrier for undifferentiated cells according to the present invention is characterized in that at least a part where cells are seeded is a ceramic having micropores having a pore diameter of 0.1 μm or more and 10 μm or less.
The micropores as described above are formed between the ceramic particles, and are present at the site where the cells are seeded so that the undifferentiated cells can easily adhere to the culture carrier and also promote the circulation of the medium. As shown, undifferentiated cells are easily formed into a three-dimensional cell cluster.
前記セラミックスは、未分化細胞の分化・誘導を起こしにくく、生体安定性、生体親和性に優れたチタニア、アルミナ、ジルコニア、イットリアおよびカーボンのうちのいずれか1種以上からなることが好ましい。 The ceramic is preferably composed of at least one of titania, alumina, zirconia, yttria, and carbon, which is unlikely to cause differentiation / induction of undifferentiated cells and is excellent in biostability and biocompatibility.
また、前記セラミックスは、内部に連通する気孔を有する多孔体からなることが好ましい。
セラミックスの内部に連通する気孔を設けることにより、培養担体内部でも培養でき、また、足場としての効果が高まるとともに、培養液が適度に循環するようになる。
なお、ここで言う「気孔」は、前記「微小孔」とは、異なるものを指す。
The ceramic is preferably made of a porous body having pores communicating with the inside.
By providing pores communicating with the inside of the ceramic, it is possible to culture inside the culture carrier, and the effect as a scaffold is enhanced, and the culture solution is circulated appropriately.
The “pores” referred to here are different from the “micropores”.
また、前記セラミックス多孔体は、無数の略球状の気孔が全体にわたって連通しており、気孔率が70%以上95%以下、平均気孔径が50μm以上1000μm以下、各気孔間の連通部の径が10μm以上200μm以下であることが好ましい。
上記のような多孔質構造により、セラミックス多孔体の表面や気孔内に、未分化細胞を効率よく撒種、定着させ、増殖させることができ、3次元細胞塊を形成した未分化細胞に、細胞増殖因子や栄養分を効率的に供給することができる。
In addition, the ceramic porous body has innumerable substantially spherical pores communicating throughout, the porosity is 70% to 95%, the average pore diameter is 50 μm to 1000 μm, and the diameter of the communication portion between each pore is It is preferable that they are 10 micrometers or more and 200 micrometers or less.
Due to the porous structure as described above, the undifferentiated cells can be efficiently seeded, fixed and proliferated on the surface and pores of the ceramic porous body. It can efficiently supply growth factors and nutrients.
また、本発明に係る未分化細胞の培養方法は、上記のような培養担体を用いて、その培養担体の表面または気孔内の少なくとも1ヶ所以上に未分化細胞を撒種して、未分化な状態で3次元細胞塊状に培養することを特徴とする。
上記のような本発明に係る培養担体を用いることにより、免疫拒絶反応を起こさず、かつ、効率的に、未分化細胞の3次元細胞塊を培養することができる。
In addition, the method for culturing undifferentiated cells according to the present invention comprises using the culture carrier as described above, seeding undifferentiated cells at least at one or more positions on the surface or pores of the culture carrier, It is characterized by culturing in a three-dimensional cell mass in a state.
By using the culture carrier according to the present invention as described above, a three-dimensional cell mass of undifferentiated cells can be efficiently cultured without causing immune rejection.
前記培養方法においては、培養担体を構成するセラミックスまたはセラミックス多孔体に直接、未分化細胞を撒種し、培養することが好ましい。
培養担体のセラミックスに、フィーダ細胞に代表される支持細胞を介さず、直接、未分化細胞を撒種(導入)させることにより、危険因子の混入や免疫拒絶反応等のおそれのない未分化細胞の3次元細胞塊を得ることができる。
In the culture method, it is preferable to seed and culture undifferentiated cells directly on ceramics or ceramic porous bodies constituting the culture carrier.
By directly seeding (introducing) undifferentiated cells into the culture carrier ceramics without supporting cells typified by feeder cells, undifferentiated cells with no risk of contamination with risk factors, immune rejection, etc. A three-dimensional cell mass can be obtained.
また、本発明に係る未分化な培養細胞は、上記のような培養担体を用いて培養され、3次元細胞塊を形成していることを特徴とする。
このような3次元細胞塊状の未分化な培養細胞は、細胞増殖因子や栄養分を付与することで、目的の組織細胞に分化・誘導することができ、複雑な組織の再生治療に応用可能になる。
Further, the undifferentiated cultured cells according to the present invention are cultured using the culture carrier as described above to form a three-dimensional cell mass.
Such undifferentiated cultured cells in the form of three-dimensional cells can be differentiated and induced into target tissue cells by applying cell growth factors and nutrients, and can be applied to regeneration treatment of complex tissues. .
また、本発明に係る未分化な培養細胞は、上記のような培養方法を用いて培養され、3次元細胞塊を形成していることを特徴とする。
このような3次元細胞塊状の未分化な培養細胞も、上記と同様に、複雑な組織の再生治療に有用である。
In addition, the undifferentiated cultured cells according to the present invention are cultured using the culture method as described above to form a three-dimensional cell mass.
Such undifferentiated cultured cells in the form of a three-dimensional cell cluster are also useful for regeneration treatment of complex tissues as described above.
上述したとおり、本発明に係る培養担体を用いれば、フィーダ細胞を用いることなく、かつ、未分化な細胞を分化・誘導させることなく、未分化な状態を保持したまま安全に培養することができる。
また、本発明によれば、セラミックスの表面や気孔内の細胞に、細胞増殖因子や栄養分を効率的に供給することができ、未分化な細胞の3次元細胞塊を形成することができる。
したがって、本発明は、ES細胞や成体多能性幹細胞等の多能性を有する未分化な細胞の培養技術の発展、ひいては、生体組織の再生治療への応用に貢献し得るものである。
As described above, when the culture carrier according to the present invention is used, it can be safely cultured while maintaining an undifferentiated state without using feeder cells and without causing differentiation / induction of undifferentiated cells. .
Further, according to the present invention, cell growth factors and nutrients can be efficiently supplied to the ceramic surface and cells in the pores, and a three-dimensional cell cluster of undifferentiated cells can be formed.
Therefore, the present invention can contribute to the development of a technique for culturing undifferentiated cells having pluripotency such as ES cells and adult pluripotent stem cells, and thus to the application to regeneration treatment of living tissue.
以下、本発明についてより詳細に説明する。
本発明に係る未分化細胞の培養担体は、少なくとも細胞を撒種する部位が、孔径0.1μm以上10μm以下の微小孔を有するセラミックスであることを特徴とするものである。
このような微小孔により、未分化細胞はセラミックスに接着しやすくなり、また、培地の循環の促進が図られ、セラミックスの表面(セラミックスが多孔体である場合には、気孔内も含む)において、未分化細胞の3次元細胞塊の形成が促進される。
Hereinafter, the present invention will be described in more detail.
The culture carrier for undifferentiated cells according to the present invention is characterized in that at least a part where cells are seeded is a ceramic having micropores having a pore diameter of 0.1 μm or more and 10 μm or less.
Such micropores make it easier for undifferentiated cells to adhere to ceramics, promote circulation of the medium, and on the surface of the ceramics (including in the pores if the ceramics are porous) Formation of a three-dimensional cell cluster of undifferentiated cells is promoted.
前記微小孔は、有機物からなる微小片の焼き抜きやセラミックス成形体の低温焼成等の方法によって、セラミックスの粒子間に形成されるものであり、部分的に存在させてもよいが、セラミックス全体に存在させることが好ましい。この微小孔を介して、3次元的に、すなわち、撒種した細胞に対してあらゆる面から培養液を供給することができる。
前記微小孔の孔径は、細胞と材料の接着に関して意味のある大きさという観点から、上記範囲内であることが好ましく、0.1μm以上5μm以下であることがより好ましい。
なお、前記微小孔の孔径は、水銀圧入法により測定することができる。
The micropores are formed between ceramic particles by a method such as burning of a small piece made of an organic material or low-temperature firing of a ceramic molded body, and may be partially present, Preferably it is present. Through this micropore, the culture solution can be supplied three-dimensionally, that is, from all sides to the seeded cells.
The pore diameter of the micropores is preferably within the above range, more preferably 0.1 μm or more and 5 μm or less, from the viewpoint of a meaningful size with respect to adhesion between cells and materials.
In addition, the hole diameter of the said micropore can be measured by the mercury intrusion method.
前記セラミックスは、チタニア、アルミナ、ジルコニア、イットリアおよびカーボンのうちのいずれか1種以上からなることが好ましい。
これらは、生体安定性、生体親和性に優れ、また、未分化細胞の分化・誘導を起こしにくい。
これらのうち、特に、チタニア、ジルコニア、イットリアが、培養された細胞が球状塊になりやすく、好ましい。
The ceramic is preferably made of at least one of titania, alumina, zirconia, yttria and carbon.
These are excellent in biostability and biocompatibility, and hardly cause differentiation / induction of undifferentiated cells.
Among these, in particular, titania, zirconia, and yttria are preferable because the cultured cells tend to be a spherical mass.
また、前記セラミックスは、内部に連通する気孔を有する多孔体からなることが好ましい。
前記微小孔と異なり、より大きな気孔が外部と連通するように存在することにより、未分化細胞が侵入し、定着できる表面積が広がり、また、培養液も3次元状に循環しやすくなる。
前記セラミックスが多孔体である場合は、少なくとも気孔内表面に、前記微小孔を有していることが好ましく、多孔体全体が微小孔を有していることがより好ましい。
なお、前記微小孔による空間は、ここでいう多孔体の気孔部分には含まれないものとする。
The ceramic is preferably made of a porous body having pores communicating with the inside.
Unlike the micropores, the presence of larger pores communicating with the outside allows the undifferentiated cells to invade, increase the surface area that can be settled, and facilitate the culture fluid to circulate three-dimensionally.
When the ceramic is a porous body, it is preferable to have the micropores at least on the pore inner surface, and it is more preferable that the entire porous body has micropores.
In addition, the space by the said micropore shall not be contained in the pore part of a porous body here.
前記セラミックス多孔体は、無数の略球状の気孔が全体にわたって連通しており、気孔率が70%以上95%以下、平均気孔径が50μm以上1000μm以下、隣り合う気孔同士の間に形成される連通部の径が10μm以上200μm以下であることが好ましい。
ただし、ここで言う平均気孔径には、前記微小孔の孔径は含まず、孔径10μmよりも大きい気孔のみを考慮した気孔径である。
また、略球状の気孔とは、厳密な真球状に限定されるものではなく、真球がやや扁平したり、歪んだりした形状等の気孔も含む意味である。
また、連通部とは、隣り合う略球状の気孔同士が接触して開口した部分をいう。開口が円形でない場合もあるが、ここでは、開口した部分の面積を有する円に置換して、その直径を「連通部の径」として表す。
The ceramic porous body has innumerable substantially spherical pores communicating with each other, and has a porosity of 70% to 95%, an average pore diameter of 50 μm to 1000 μm, and a communication formed between adjacent pores. The diameter of the part is preferably 10 μm or more and 200 μm or less.
However, the average pore diameter referred to here does not include the pore diameter of the micropores, and is a pore diameter considering only pores larger than the pore diameter of 10 μm.
Further, the substantially spherical pore is not limited to a strictly spherical shape, but includes a pore having a shape in which the true sphere is slightly flattened or distorted.
Moreover, a communicating part means the part which adjacent substantially spherical pores contacted and opened. In some cases, the opening is not circular, but here, the opening is replaced with a circle having the area of the opening, and the diameter is expressed as “the diameter of the communication portion”.
上記のような構造を有するセラミックス多孔体によれば、該多孔体の孔内に、未分化細胞を効率よく撒種、定着させ、セラミックス多孔体上で増殖させることができ、かつ、3次元細胞塊を形成した未分化細胞に、細胞増殖因子や栄養分を効率的に供給することができる。
なお、前記気孔率は、多孔体の密度と理論密度から導くことができる。また、平均気孔径は、特許第3400740号公報に記載の樹脂包埋による方法で求められ、連通部の径は、水銀圧入法により求めることができる。
According to the ceramic porous body having the structure as described above, undifferentiated cells can be efficiently seeded and fixed in the pores of the porous body, and can be propagated on the ceramic porous body. Cell growth factors and nutrients can be efficiently supplied to undifferentiated cells that have formed clumps.
The porosity can be derived from the density and theoretical density of the porous body. The average pore diameter can be obtained by the resin embedding method described in Japanese Patent No. 3400740, and the diameter of the communicating portion can be obtained by the mercury intrusion method.
前記セラミックス多孔体の気孔率が70%未満である場合、また、平均気孔径が50μm未満である場合、未分化細胞や培養液が容易に侵入できるような連通部の径を得ることが難しい。
一方、前記気孔率が95%を超える場合、また、平均気孔径が1000μmを超える場合、多孔体内部に侵入した未分化細胞が流出しやすく、多孔体上に定着し難くなるとともに、培養担体の形状を保つことが難しい。
前記気孔率は、より好ましくは、75%以上90%以下であり、さらに、80%以上90%以下であることが好ましい。また、平均気孔径は、250μm以上800μm以下であることがより好ましい。
When the porosity of the ceramic porous body is less than 70%, and when the average pore diameter is less than 50 μm, it is difficult to obtain the diameter of the communication part that allows undifferentiated cells and culture medium to easily enter.
On the other hand, when the porosity exceeds 95% and the average pore diameter exceeds 1000 μm, undifferentiated cells that have entered the porous body are likely to flow out and are difficult to settle on the porous body. It is difficult to keep the shape.
The porosity is more preferably 75% or more and 90% or less, and further preferably 80% or more and 90% or less. Further, the average pore diameter is more preferably 250 μm or more and 800 μm or less.
また、前記セラミックス多孔体の各気孔間の連通部の径が10μm未満である場合、細胞を培養する担体の連通性が不十分であり、培地に供給した細胞増殖因子や栄養分等が、十分に細胞に行き渡らず、担体内部の細胞が死滅したり、目的の細胞に分化しないおそれがある。
一方、前記径が200μmを超える場合、多孔体内部に侵入した未分化細胞が流出しやすく、多孔体上に定着し難くなる。
前記連通部の径は、20μm以上150μm以下であることがより好ましい。
なお、上記のようなセラミックス多孔体は、スポンジ状の有機多孔体にスラリーを塗布して有機多孔体を焼き抜くセラミックフォームや、スラリーを撹拌起泡して焼結する特許第3400740号に記載されている方法等によって得ることができるが、気孔の制御がしやすい後者の方法により得ることがより好ましい。
Moreover, when the diameter of the communicating portion between the pores of the ceramic porous body is less than 10 μm, the connectivity of the carrier for culturing the cells is insufficient, and the cell growth factor and nutrients supplied to the medium are sufficiently There is a risk that the cells inside the carrier will not die or will not differentiate into the target cells.
On the other hand, when the diameter exceeds 200 μm, undifferentiated cells that have entered the porous body easily flow out, and are difficult to settle on the porous body.
The diameter of the communicating part is more preferably 20 μm or more and 150 μm or less.
The ceramic porous body as described above is described in ceramic foam in which a slurry is applied to a sponge-like organic porous body to burn out the organic porous body, or in Japanese Patent No. 3400740 in which the slurry is stirred and foamed and sintered. However, it is more preferable to obtain by the latter method in which the pores are easily controlled.
本発明に係る培養担体を適用する細胞は、ES細胞等に代表される未分化細胞である。未分化細胞とは、自分自身と同じ細胞に増殖する能力と、細胞増殖因子の付与によって決まった組織細胞に分化する能力を有する未分化の状態の細胞であり、一旦、他の組織細胞に分化すると、未分化な状態に戻ることはできない。具体的には、胚性幹細胞(ES細胞)、間葉系幹細胞、造血幹細胞、神経幹細胞、肝臓幹細胞、膵臓幹細胞、皮膚幹細胞等が挙げられるが、本発明においては、ES細胞や間葉系幹細胞を用いることが好ましく、特に、ES細胞を用いることが好ましい。 The cells to which the culture carrier according to the present invention is applied are undifferentiated cells represented by ES cells and the like. An undifferentiated cell is an undifferentiated cell that has the ability to grow into the same cell as itself and the ability to differentiate into a tissue cell determined by the addition of a cell growth factor. Once differentiated into another tissue cell Then, it cannot return to an undifferentiated state. Specific examples include embryonic stem cells (ES cells), mesenchymal stem cells, hematopoietic stem cells, neural stem cells, liver stem cells, pancreatic stem cells, skin stem cells, etc. In the present invention, ES cells and mesenchymal stem cells are exemplified. It is preferable to use ES cells, and it is particularly preferable to use ES cells.
また、上記のような細胞の培養に用いられる培地は、特に限定されるものではなく、培養する細胞に応じて、適宜選択することができる。例えば、MEM、α−MEM、DMEM、イーグル培地等が好適に用いられる。
これらの培地には、さらに、FBS(fetal bovine serum;ウシ胎児血清)、KSR(KnockOutTM Serum Replacement)、LIF(leukemia inhibitory factor;白血病阻害因子)、非必須アミノ酸、ピルビン酸、抗生物質等の細胞を維持するために必要な物質を添加することが好ましい。
The medium used for culturing the cells as described above is not particularly limited, and can be appropriately selected depending on the cells to be cultured. For example, MEM, α-MEM, DMEM, Eagle medium, etc. are preferably used.
These media also contain cells such as FBS (fetal bovine serum), KSR (KnockOut ™ Serum Replacement), LIF (leukemia inhibitory factor), non-essential amino acids, pyruvate, antibiotics, etc. It is preferable to add a substance necessary for maintaining the temperature.
さらにまた、セラミックスの表面や多孔体の気孔内部で未分化な状態で形成された3次元細胞塊を、目的とする細胞に分化・誘導するために、例えば、骨となる骨芽細胞に誘導するためには、FGF(線維芽細胞成長因子)、IGF−1、IGF−II、PDGF(血小板由来成長因子)、TGF−B(トランスフォーミング成長因子)、BMP−Z、HGH、ヒト由来成長因子の濃縮物等の細胞増殖因子を培地に添加してもよい。 Furthermore, in order to differentiate and induce a three-dimensional cell cluster formed in an undifferentiated state on the ceramic surface or inside the pores of the porous body, for example, it is induced to osteoblasts that become bones. For this purpose, FGF (fibroblast growth factor), IGF-1, IGF-II, PDGF (platelet-derived growth factor), TGF-B (transforming growth factor), BMP-Z, HGH, human-derived growth factor Cell growth factors such as concentrates may be added to the medium.
上記のような本発明に係る培養担体を用いて、前記セラミックス(培養担体)の表面または気孔内の少なくとも1ヶ所以上に未分化細胞を撒種し、適宜、細胞増殖因子や栄養分を培地に添加して供給することにより、未分化な状態を保持したままで、3次元細胞塊に形成された未分化な培養細胞を得ることができる。
このような3次元細胞塊状の未分化な培養細胞は、複雑な組織の再生治療に有効活用することができる。
Using the culture carrier according to the present invention as described above, undifferentiated cells are seeded on at least one surface of the ceramics (culture carrier) or in the pores, and cell growth factors and nutrients are appropriately added to the medium. Thus, undifferentiated cultured cells formed into a three-dimensional cell mass can be obtained while maintaining an undifferentiated state.
Such undifferentiated cultured cells in the form of three-dimensional cells can be effectively used for regeneration treatment of complex tissues.
特に、本発明に係る培養担体を用いれば、培養担体であるセラミックス上に、フィーダ細胞層を形成することなく、直接未分化細胞を撒種することにより、純粋な未分化な培養細胞を容易に得ることができるという利点を有している。
また、前記培養細胞は、図3−2の電子顕微鏡写真に見られるような球状の細胞塊として得ることもできる(下記実施例3参照)。
In particular, when the culture carrier according to the present invention is used, pure undifferentiated cultured cells can be easily obtained by seeding undifferentiated cells directly on the ceramic as a culture carrier without forming a feeder cell layer. It has the advantage that it can be obtained.
The cultured cells can also be obtained as a spherical cell mass as seen in the electron micrograph of FIG. 3-2 (see Example 3 below).
以下、本発明を実施例に基づいてさらに具体的に説明するが、本発明は、下記実施例により制限されるものではない。
[実施例1](チタニアセラミックス多孔体(焼結体)の作製)
セラミックス原料として平均粒径180nmのチタニア粉末810gと、分散剤としてポリカルボン酸アンモニウム塩0.41gとポリエチレンイミン32.4gを、分散溶媒として純水270gを、ボールミルで15時間撹拌混合して原料スラリーを調製した。
この原料スラリーに、起泡剤としてエマール(登録商標)4.8gを添加して撹拌し、泡沫状スラリーとした。
さらに、ゲル化剤としてソルビトールポリグリシジルエーテル4.2gを加えて、150mm×150mm×30mmの型に流し込み、加湿乾燥させ、チタニア多孔体の成形体を得た。
この成形体を、1200℃で2時間焼成して、135mm×134mm×21mmのチタニアセラミックス多孔体(焼結体)を得た。
この焼結体の気孔率は84.8%であった。
得られた焼結体の電子顕微鏡写真(10000倍、100倍)を、図1−1、図1−2に示す。
図1−1に示す電子顕微鏡写真から、チタニアセラミックス多孔体の骨格部分に、孔径0.5〜2μmの微小孔が存在することが観察され、これを水銀圧入法により測定したところ、孔径0.4〜1.0μmであった。また、平均気孔径は150〜450μm、連通部の径は40〜60μmであった。
EXAMPLES Hereinafter, although this invention is demonstrated further more concretely based on an Example, this invention is not restrict | limited by the following Example.
[Example 1] (Preparation of titania ceramic porous body (sintered body))
810 g of titania powder having an average particle diameter of 180 nm as a ceramic raw material, 0.41 g of polycarboxylic acid ammonium salt and 32.4 g of polyethyleneimine as a dispersing agent, 270 g of pure water as a dispersing solvent, and stirring and mixing for 15 hours in a ball mill Was prepared.
To this raw material slurry, 4.8 g of Emar (registered trademark) as a foaming agent was added and stirred to obtain a foamy slurry.
Furthermore, 4.2 g of sorbitol polyglycidyl ether was added as a gelling agent, poured into a 150 mm × 150 mm × 30 mm mold, dried by humidification, and a titania porous body molded body was obtained.
This molded body was fired at 1200 ° C. for 2 hours to obtain a titania ceramic porous body (sintered body) of 135 mm × 134 mm × 21 mm.
The porosity of this sintered body was 84.8%.
Electron micrographs (10000 times, 100 times) of the obtained sintered body are shown in FIGS. 1-1 and 1-2.
From the electron micrograph shown in FIG. 1-1, it was observed that micropores having a pore diameter of 0.5 to 2 μm were present in the skeletal portion of the titania ceramic porous body, and this was measured by a mercury intrusion method. It was 4 to 1.0 μm. Moreover, the average pore diameter was 150 to 450 μm, and the diameter of the communicating part was 40 to 60 μm.
[実施例2]
実施例1と同様にして得られたチタニア多孔体の成形体を、1400℃で2時間焼成して、135mm×134mm×21mmのチタニアセラミックス多孔体(焼結体)を得た。
この焼結体の気孔率は80.2%であった。
得られた焼結体の電子顕微鏡写真(10000倍)を、図2に示す。また、平均気孔径は150〜450μm、連通部の径は40〜60μmであった。
図2に示す電子顕微鏡写真においては、図1−1で見られたような微小孔は消失している。
[Example 2]
The formed titania porous body obtained in the same manner as in Example 1 was fired at 1400 ° C. for 2 hours to obtain a 135 mm × 134 mm × 21 mm titania ceramic porous body (sintered body).
The porosity of this sintered body was 80.2%.
An electron micrograph (10,000 times) of the obtained sintered body is shown in FIG. Moreover, the average pore diameter was 150 to 450 μm, and the diameter of the communicating part was 40 to 60 μm.
In the electron micrograph shown in FIG. 2, the micropores as seen in FIG. 1-1 have disappeared.
[実施例3](チタニアセラミックス多孔体上でのES細胞培養)
96穴プレートの穴に、実施例1で作製したチタニアセラミックス多孔体を径5mm高さ2mmの円柱状に加工して充填し、このチタニアセラミックス多孔体上に、予め培養されたES細胞を5.0×105個撒種し、KSR、ピルビン酸、非必須アミノ酸、LIF、ストレプトマイシン、ペニシリンを含んだDMEMにおいて、5%CO2インキュベータ内で、37℃で3日間培養した。
培養後の細胞の電子顕微鏡写真(100倍、1000倍)を、図3−1、3−2に示す。
図3−2に示す電子顕微鏡写真から、チタニアセラミックス多孔体上で、ES細胞が未分化な状態を保持したまま、3次元細胞塊を形成していることが確認された。さらに、3次元細胞塊をALP(アルカリフォスターゼ)染色したところ、3次元細胞塊が青紫色に染色され、ES細胞が未分化な状態を保持したまま、増殖していることが確認された。
[Example 3] (ES cell culture on titania ceramic porous body)
The 96-well plate was filled with the titania ceramic porous body produced in Example 1 in a cylindrical shape with a diameter of 5 mm and a height of 2 mm, and ES cells cultured in advance on the porous titania ceramic body were filled with 5. 0 × 10 5 seeded and cultured in DMEM containing KSR, pyruvate, non-essential amino acids, LIF, streptomycin and penicillin at 37 ° C. for 3 days in a 5% CO 2 incubator.
Electron micrographs (100 times and 1000 times) of the cultured cells are shown in FIGS.
From the electron micrograph shown in FIG. 3-2, it was confirmed that the ES cells formed a three-dimensional cell mass on the titania ceramic porous body while maintaining an undifferentiated state. Further, when the three-dimensional cell mass was stained with ALP (alkaline phosphatase), the three-dimensional cell mass was stained blue-purple, and it was confirmed that the ES cells were proliferating while maintaining an undifferentiated state.
[実施例4](ハイドロキシアパタイトセラミックス上でのES細胞培養)
チタニアセラミックス多孔体に代えて、ハイドロキシアパタイトセラミックス多孔体(気孔率80%)を用いて、実施例3と同様にして、ES細胞を培養した。
培養後の細胞の電子顕微鏡写真(1000倍)を、図4に示す。
図4に示す電子顕微鏡写真から、ハイドロキシアパタイトセラミックス上では、ES細胞の3次元細胞塊の形成は認められなかった。
[Example 4] (ES cell culture on hydroxyapatite ceramics)
ES cells were cultured in the same manner as in Example 3 using a hydroxyapatite ceramic porous body (porosity 80%) instead of the titania ceramic porous body.
The electron micrograph (1000 times) of the cell after culture | cultivation is shown in FIG.
From the electron micrograph shown in FIG. 4, formation of a three-dimensional cell cluster of ES cells was not observed on the hydroxyapatite ceramics.
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