JP2010000020A - Carrier for cell culture - Google Patents
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- JP2010000020A JP2010000020A JP2008160473A JP2008160473A JP2010000020A JP 2010000020 A JP2010000020 A JP 2010000020A JP 2008160473 A JP2008160473 A JP 2008160473A JP 2008160473 A JP2008160473 A JP 2008160473A JP 2010000020 A JP2010000020 A JP 2010000020A
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- 238000004113 cell culture Methods 0.000 title claims abstract description 29
- 239000000758 substrate Substances 0.000 claims abstract description 11
- 239000000463 material Substances 0.000 claims description 8
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 6
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 claims description 6
- MCMNRKCIXSYSNV-UHFFFAOYSA-N Zirconium dioxide Chemical compound O=[Zr]=O MCMNRKCIXSYSNV-UHFFFAOYSA-N 0.000 claims description 6
- -1 polyethylene Polymers 0.000 claims description 6
- 108010010803 Gelatin Proteins 0.000 claims description 5
- 229920000159 gelatin Polymers 0.000 claims description 5
- 239000008273 gelatin Substances 0.000 claims description 5
- 235000019322 gelatine Nutrition 0.000 claims description 5
- 235000011852 gelatine desserts Nutrition 0.000 claims description 5
- 229910052588 hydroxylapatite Inorganic materials 0.000 claims description 5
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 claims description 5
- 229920000642 polymer Polymers 0.000 claims description 4
- 229920000178 Acrylic resin Polymers 0.000 claims description 3
- 239000004925 Acrylic resin Substances 0.000 claims description 3
- 229920000936 Agarose Polymers 0.000 claims description 3
- 102000008186 Collagen Human genes 0.000 claims description 3
- 108010035532 Collagen Proteins 0.000 claims description 3
- 239000004698 Polyethylene Substances 0.000 claims description 3
- 239000004743 Polypropylene Substances 0.000 claims description 3
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 claims description 3
- 229920001436 collagen Polymers 0.000 claims description 3
- 229920000747 poly(lactic acid) Polymers 0.000 claims description 3
- 229920000573 polyethylene Polymers 0.000 claims description 3
- 239000004626 polylactic acid Substances 0.000 claims description 3
- 229920001155 polypropylene Polymers 0.000 claims description 3
- 239000000377 silicon dioxide Substances 0.000 claims description 3
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 claims description 3
- RUDFQVOCFDJEEF-UHFFFAOYSA-N yttrium(III) oxide Inorganic materials [O-2].[O-2].[O-2].[Y+3].[Y+3] RUDFQVOCFDJEEF-UHFFFAOYSA-N 0.000 claims description 3
- 239000000919 ceramic Substances 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 abstract description 71
- 238000000338 in vitro Methods 0.000 abstract description 8
- 210000004748 cultured cell Anatomy 0.000 abstract description 4
- 210000001519 tissue Anatomy 0.000 description 5
- 239000003814 drug Substances 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 238000010899 nucleation Methods 0.000 description 4
- 230000005540 biological transmission Effects 0.000 description 3
- 230000021164 cell adhesion Effects 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 238000001000 micrograph Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 108010088751 Albumins Proteins 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 2
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 2
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- 238000012136 culture method Methods 0.000 description 2
- 210000002744 extracellular matrix Anatomy 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
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- 206010073071 hepatocellular carcinoma Diseases 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
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- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 230000004931 aggregating effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000012832 cell culture technique Methods 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
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- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
【課題】生体組織から採取した細胞の生体外培養において、生体内により近い環境下で、三次元的な細胞凝集体を簡易に形成することができ、かつ、培養面積当たりの培養細胞数を増加させることができる細胞培養担体を提供する。
【解決手段】基材表面２に、幅が１０μｍ以上１００００μｍ以下、深さが１０μｍ以上１００００μｍ以下であり、端部が閉じられた、凹曲面状の溝１が、平行ライン状に複数形成されている細胞培養担体を用いる。
【選択図】図１In an in vitro culture of cells collected from a living tissue, a three-dimensional cell aggregate can be easily formed in an environment closer to the living body, and the number of cultured cells per culture area can be increased. Provided is a cell culture carrier that can be prepared.
SOLUTION: A plurality of concave curved grooves 1 having a width of 10 μm or more and 10000 μm or less, a depth of 10 μm or more and 10000 μm or less, and closed ends are formed on a substrate surface 2 in a parallel line shape. Cell culture carrier.
[Selection] Figure 1
Description
本発明は、生体外で、三次元的な細胞凝集体を形成し、細胞の機能や寿命を維持して増殖させるための細胞培養担体に関する。 The present invention relates to a cell culture carrier for forming a three-dimensional cell aggregate in vitro and maintaining the function and life of the cell to proliferate.
近年、ＥＳ細胞の樹立や、生体組織から採取した幹細胞や組織細胞を選別する技術の発達等により、これらの細胞を生体外で培養し、細胞治療への応用や、薬剤の生理活性・毒性等を調べる薬物シミュレータに応用する研究が盛んに行われている。 In recent years, with the establishment of ES cells and the development of techniques for selecting stem cells and tissue cells collected from living tissues, these cells are cultured in vitro to apply to cell therapy, physiological activity / toxicity of drugs, etc. Research that applies to drug simulators is being actively conducted.
一般に、生体内では、細胞が組織化する際に、細胞の機能の維持向上を図る上で重要なファクターである細胞外マトリックスや内部圧力等について、細胞同士間での情報伝達を活発に行うための環境が整っている。
しかしながら、生体外では、このような生体内と同様の環境を作ることが難しいため、生体から採取した細胞の生体外培養は、非常に困難である。
このため、生体外培養において、できる限り生体内に近い環境下で、細胞を培養する技術の開発が求められている。
In general, in order to actively communicate information between cells about extracellular matrix and internal pressure, which are important factors for maintaining and improving cell functions when cells are organized in vivo. The environment is in place.
However, since it is difficult to create an environment similar to that in the living body outside the living body, it is very difficult to culture the cells collected from the living body in vitro.
For this reason, in vitro culture, development of a technique for culturing cells in an environment as close as possible to the living body is required.
これに対して、細胞培養技術の発達に伴い、最近では、生体組織から採取した細胞を、その細胞に適した細胞外マトリックスをコーティングしたディッシュ上で維持する技術が知られている。
しかしながら、ディッシュ上で増殖した細胞は、二次元的にしか増殖することができないため、細胞の寿命や物質生産能力等が、生体内とは異なっていることが多い。
On the other hand, with the development of cell culture techniques, recently, a technique for maintaining cells collected from a living tissue on a dish coated with an extracellular matrix suitable for the cells is known.
However, since the cells grown on the dish can grow only two-dimensionally, the lifespan of the cells, the substance production ability, etc. are often different from those in the living body.
このため、細胞間相互作用の維持の観点から、多数の細胞から三次元的な凝集体を作製するスフェロイド（球状細胞凝集体）培養法が注目されている。
例えば、その一つとして、細胞非接着性材料からなる基板表面に、一定の間隔で、直径数十〜数百μｍの細胞接着性を有する半球状のマイクロウェルを配列させた培養担体を用い、このウェル（凹部）内に接着依存性細胞を大量に撒種することにより、ウェル内で凝集した細胞同士が接着し、細胞凝集体が形成されるという培養方法が知られている（例えば、特許文献１）。
For this reason, a spheroid (spherical cell aggregate) culture method for producing a three-dimensional aggregate from a large number of cells has attracted attention from the viewpoint of maintaining the interaction between cells.
For example, as one of them, a culture carrier in which hemispherical microwells having a cell adhesion of several tens to several hundreds μm in diameter are arranged on a substrate surface made of a cell non-adhesive material at regular intervals, A culture method is known in which a large number of adhesion-dependent cells are seeded in the wells (recesses) so that cells aggregated in the wells adhere to each other to form cell aggregates (for example, patents). Reference 1).
しかしながら、上記のような従来の培養担体を用いた場合、培養面積当たりのスフェロイド形成効率は十分ではない。また、各マイクロウェル同士は連結していないため、マイクロウェル内で形成された細胞凝集体同士の情報伝達はなされず、培養細胞のための環境が、人工臓器や薬物シミュレータへの応用に適しているとは言い難い。 However, when the conventional culture carrier as described above is used, the spheroid formation efficiency per culture area is not sufficient. In addition, since the microwells are not connected to each other, information transmission between the cell aggregates formed in the microwells is not performed, and the environment for cultured cells is suitable for application to artificial organs and drug simulators. It ’s hard to say.
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、生体組織から採取した細胞の生体外培養において、生体内により近い環境下で、三次元的な細胞凝集体を簡易に形成することができ、かつ、培養面積当たりの細胞数を増加させることができる細胞培養担体を提供することを目的とするものである。 The present invention has been made to solve the above technical problem, and in the in vitro culture of cells collected from a living tissue, a three-dimensional cell aggregate is easily formed in an environment closer to the living body. It is an object of the present invention to provide a cell culture carrier capable of increasing the number of cells per culture area.
本発明に係る細胞培養担体は、基材表面に、平行ライン状に、端部が閉じられた溝が複数形成されていることを特徴とする。
このような培養担体を用いることにより、一つの溝内で、複数の細胞同士間での情報伝達が可能となり、生体内に近い環境下で、三次元状の細胞凝集体を大量に培養することができる。
The cell culture carrier according to the present invention is characterized in that a plurality of grooves whose ends are closed are formed in parallel lines on the surface of the substrate.
By using such a culture carrier, information can be transmitted between multiple cells in a single groove, and a large amount of three-dimensional cell aggregates can be cultured in an environment close to the living body. Can do.
また、本発明に係る他の態様の細胞培養担体は、基材表面に、網目状に、端部が閉じられた溝が複数形成されていることを特徴とする。
このような培養担体を用いれば、溝内での、複数の細胞同士間での情報伝達がさらに密になり、より生体内に近い環境が得られる。
Moreover, the cell culture carrier according to another aspect of the present invention is characterized in that a plurality of grooves whose ends are closed are formed in a mesh shape on the surface of the base material.
If such a culture carrier is used, information transmission between a plurality of cells in the groove becomes more dense, and an environment closer to the living body can be obtained.
前記溝は、培養担体に撒種した細胞を凝集させてスフェロイド化させる観点から、凹曲面状であることが好ましい。 The groove is preferably concavely curved from the viewpoint of aggregating cells seeded on the culture carrier to spheroidize.
また、前記溝は、培養された三次元状の細胞凝集体の取り扱い容易等の観点から、幅が１０μｍ以上１００００μｍ以下、深さが１０μｍ以上１００００μｍ以下であることが好ましい。 In addition, the groove preferably has a width of 10 μm or more and 10,000 μm or less and a depth of 10 μm or more and 10,000 μm or less from the viewpoint of easy handling of the cultured three-dimensional cell aggregate.
さらに、前記溝は、撒種する細胞の位置を固定させる観点から、底部より深い凹部が等間隔に形成されていることが好ましい。 Furthermore, it is preferable that the groove has recesses deeper than the bottom formed at equal intervals from the viewpoint of fixing the position of cells to be seeded.
また、前記基材は、細胞の接着性や増殖効率等に応じて選択され、具体的には、ハイドロキシアパタイト、β−リン酸三カルシウム、アルミナ、ジルコニア、チタニア、シリカおよびイットリアのうちの少なくともいずれか１種のセラミックス、または、ポリエチレン、ポリプロピレン、アクリル樹脂、アガロース、コラーゲン、ゼラチンもしくはポリ乳酸のうちの少なくともいずれか１種の高分子からなることが好ましい。 Further, the substrate is selected according to cell adhesion, proliferation efficiency, etc., specifically, at least any one of hydroxyapatite, β-tricalcium phosphate, alumina, zirconia, titania, silica, and yttria. Preferably, the polymer is made of at least one kind of polymer selected from polyethylene, polypropylene, acrylic resin, agarose, collagen, gelatin, and polylactic acid.
本発明に係る細胞培養担体を用いれば、生体組織から採取した細胞の生体外培養において、細胞の機能や寿命を維持した三次元的な細胞凝集体を簡易に形成し、かつ、培養面積当たりの培養細胞数を増加させることができる。
したがって、本発明に係る細胞培養担体によれば、生体外培養で、人工臓器や薬物シミュレータへの応用にも適した培養細胞を提供することができる。
With the use of the cell culture carrier according to the present invention, in vitro culture of cells collected from living tissue, three-dimensional cell aggregates that maintain the function and life of the cells can be easily formed, and per cell area The number of cultured cells can be increased.
Therefore, according to the cell culture carrier according to the present invention, cultured cells suitable for application to artificial organs and drug simulators can be provided by in vitro culture.
以下、本発明について、図面を参照して、より詳細に説明する。
図１に、本発明に係る細胞培養担体の一態様の上面図を、図２に、その顕微鏡写真を、また、図３に、その部分斜視図を示す。
この細胞培養担体は、円板状の基材表面２に、平行ライン状に、溝１が複数形成されている。各溝１は、端部が閉じられた状態であり、上面から見ると、細長い長方形状である。
このような形状の培養担体を用いて、一つの溝１内に間隔をあけて複数の細胞を撒種することにより、従来のマイクロウェルが形成された培養担体よりも広面積である溝１内において、三次元状の細胞凝集体を大量に培養することができる。
また、一つの溝１で、複数の細胞の培養が行われることにより、細胞同士間での情報伝達が可能となり、独立したウェル毎に細胞が培養される場合よりも、生体内に近い環境を形成することができるため、細胞の寿命や物質生産能力の向上を図ることができる。
Hereinafter, the present invention will be described in more detail with reference to the drawings.
FIG. 1 is a top view of one embodiment of the cell culture carrier according to the present invention, FIG. 2 is a micrograph thereof, and FIG. 3 is a partial perspective view thereof.
In this cell culture carrier, a plurality of grooves 1 are formed in a parallel line shape on a disk-like substrate surface 2. Each groove 1 is in a state in which the end is closed, and has an elongated rectangular shape when viewed from above.
By using a culture carrier having such a shape, a plurality of cells are seeded at intervals in one groove 1 so that the inside of the groove 1 has a larger area than the culture carrier on which a conventional microwell is formed. In, three-dimensional cell aggregates can be cultured in large quantities.
In addition, by culturing a plurality of cells in one groove 1, it becomes possible to transmit information between cells, and an environment closer to the living body than when cells are cultured for each independent well. Since it can form, the lifetime of a cell and the improvement of a substance production capacity can be aimed at.
培養させる三次元状の細胞凝集体の大きさの制御は、端部が閉じられた溝１の長手方向（ライン方向）の長さを制御することにより行うことができる。
前記各溝１の端部が閉じられていない場合、細胞の播種時に、細胞が端部より流れ落ちるため、培養させる三次元状の細胞凝集体の大きさを制御することができない。
The size of the three-dimensional cell aggregate to be cultured can be controlled by controlling the length in the longitudinal direction (line direction) of the groove 1 whose end is closed.
When the ends of the grooves 1 are not closed, the cells flow down from the ends when the cells are seeded, and thus the size of the three-dimensional cell aggregate to be cultured cannot be controlled.
前記溝１は、図３に示すように、凹曲面状（Ｕ字状）であることが好ましい。また、断面が半円状であることがより好ましい。
該溝１内に撒種された細胞を凝集させてスフェロイド化させる観点から、このように、溝１は角部を有しない形状が適している。
As shown in FIG. 3, the groove 1 preferably has a concave curved surface shape (U-shape). More preferably, the cross section is semicircular.
From the viewpoint of agglomerating the cells seeded in the groove 1 to form a spheroid, the groove 1 is suitable in a shape having no corners.
また、前記溝１のサイズは、培養する細胞の大きさに応じて、適宜設計することができるが、培養された三次元状の細胞凝集体の取り扱い容易等の観点から、幅および深さは１０μｍ以上１００００μｍ以下であることが好ましい。 The size of the groove 1 can be appropriately designed according to the size of the cells to be cultured. However, from the viewpoint of easy handling of the cultured three-dimensional cell aggregates, the width and depth are It is preferable that they are 10 micrometers or more and 10,000 micrometers or less.
なお、前記溝１の間隔、すなわち、溝１同士の間に露出している基板表面（凸部）２の幅は、５μｍ以上１００μｍ以下であることが好ましい。
凸部は、細胞非接着部分であり、培養担体上での培養面積を広げる観点から、できる限り面積が小さいことが好ましいが、培養担体の強度を考慮して、上記範囲内の幅とすることが好ましい。凸部２の幅は、より好ましくは、５０μｍ程度である。
In addition, it is preferable that the space | interval of the said groove | channel 1, ie, the width | variety of the substrate surface (convex part) 2 exposed between the groove | channels 1, is 5 micrometers or more and 100 micrometers or less.
The convex part is a cell non-adhering part, and it is preferable that the area is as small as possible from the viewpoint of expanding the culture area on the culture carrier, but the width should be within the above range in consideration of the strength of the culture carrier. Is preferred. More preferably, the width of the protrusion 2 is about 50 μm.
図４に、本発明に係る細胞培養担体の他の態様の部分斜視図を示す。また、図５に、図４のＡ−Ａ断面図を示す。
図４は、細胞培養担体全体は、上記図１示したものと同様に、円板状の基材表面２に、平行ライン状に、端部が閉じられた状態で、溝１が複数形成されているものである。上記の態様とは、溝１の形状のみが異なっている。
FIG. 4 shows a partial perspective view of another embodiment of the cell culture carrier according to the present invention. FIG. 5 is a cross-sectional view taken along the line AA in FIG.
FIG. 4 shows that the entire cell culture carrier is formed with a plurality of grooves 1 on the disk-like base material surface 2 in the form of parallel lines with the ends closed in the same manner as shown in FIG. It is what. Only the shape of the groove 1 is different from the above aspect.
本態様は、図４，５に示したように、溝１のライン方向において、溝１の底部より深い凹部３が等間隔に形成されているものである。前記凹部３の深さ（溝１の底部からの深さ）は、前記撒種させる細胞の大きさよりも小さく構成されている。
このように、凹部３を予め溝１の底部に形成し、その位置に細胞を撒種させることにより、撒種させた直後の細胞の位置ずれを防止することができる。これにより、撒種直後の細胞同士の衝突を防止することができ、細胞へのダメージを防止することができる。
前記凹部３の深さが、前記撒種させる細胞の大きさよりも大きい場合、前記凹部３内のみで細胞が培養される可能性があり、上述した効果が十分に得られない。
この凹部３も、図５に示すように、上述した溝１の形状と同様に、細胞のスフェロイド化の観点から、角部を有しないように、凹曲面状に形成されていることが好ましい。
In this embodiment, as shown in FIGS. 4 and 5, recesses 3 deeper than the bottom of the groove 1 are formed at equal intervals in the line direction of the groove 1. The depth of the recess 3 (depth from the bottom of the groove 1) is configured to be smaller than the size of the cell to be seeded.
In this way, by forming the recess 3 in the bottom of the groove 1 in advance and seeding the cell at that position, it is possible to prevent the displacement of the cell immediately after seeding. Thereby, the collision of the cells immediately after seeding can be prevented, and damage to the cells can be prevented.
When the depth of the concave portion 3 is larger than the size of the cell to be seeded, the cells may be cultured only in the concave portion 3, and the above-described effects cannot be sufficiently obtained.
As shown in FIG. 5, the concave portion 3 is preferably formed in a concave curved surface shape so as not to have a corner portion from the viewpoint of spheroidization of cells, similarly to the shape of the groove 1 described above.
また、図６に、本発明に係る細胞培養担体の他の態様を示す。図６に示す培養担体は、上記の図１に示した培養担体における溝１が、基板表面２に、平行ライン状のみならず、直交ライン状にも複数形成されているものである。すなわち、端部が閉じられた溝１が、網目状に形成されているものである。
各溝１の形状、サイズ等は、上記図１に示した培養担体と同様であるため、説明を省略する。
このように、網目状に溝１を形成することにより、溝１内に撒種された細胞同士間での情報伝達がさらに密になり、より生体内に近い環境を形成することができる。
FIG. 6 shows another embodiment of the cell culture carrier according to the present invention. In the culture carrier shown in FIG. 6, a plurality of grooves 1 in the culture carrier shown in FIG. 1 are formed on the substrate surface 2 not only in the form of parallel lines but also in the form of orthogonal lines. That is, the groove | channel 1 with which the edge part was closed is formed in mesh shape.
The shape, size, etc. of each groove 1 are the same as those of the culture carrier shown in FIG.
Thus, by forming the grooves 1 in a mesh shape, information transmission between cells seeded in the grooves 1 becomes more dense, and an environment closer to the living body can be formed.
上記図６に示したような網目状の溝１も、図４，５に示すような底部がより深い凹部を等間隔に形成しておいてもよい。この場合、前記凹部は、直交する溝１の各交点の位置に形成されることが好ましい。 The mesh-shaped grooves 1 as shown in FIG. 6 may also have recesses with deeper bottoms as shown in FIGS. 4 and 5 formed at equal intervals. In this case, it is preferable that the concave portion is formed at each intersection of the orthogonal grooves 1.
本発明に係る細胞培養担体の基材の材質は、細胞の接着性や増殖効率等に応じて、従来から培養担体に用いられている材料のうちから、適宜選択することが好ましい。具体的には、ハイドロキシアパタイト、β−リン酸三カルシウム、アルミナ、ジルコニア、チタニア、シリカおよびイットリアのうちの少なくともいずれか１種のセラミックス、または、ポリエチレン、ポリプロピレン、アクリル樹脂、アガロース、コラーゲン、ゼラチンもしくはポリ乳酸のうちの少なくともいずれか１種の高分子からなることが好ましい。 The material of the base material of the cell culture carrier according to the present invention is preferably selected appropriately from materials conventionally used for culture carriers according to cell adhesion, proliferation efficiency and the like. Specifically, at least one ceramic selected from hydroxyapatite, β-tricalcium phosphate, alumina, zirconia, titania, silica and yttria, or polyethylene, polypropylene, acrylic resin, agarose, collagen, gelatin or It is preferably made of at least any one polymer of polylactic acid.
なお、前記溝１の内表面は、細胞を効率的に接着させる観点から、多孔体であることが好ましい。ここでいう多孔体とは、例えば、気孔率が３０〜９５％である。
ただし、培養担体に対する接着性が強い細胞を培養する場合は、逆に、接着性を抑制する必要があるため、前記溝１の内表面は緻密体であることが好ましい。ここでいう緻密体とは、例えば、気孔率が９５％以上である。
The inner surface of the groove 1 is preferably a porous body from the viewpoint of efficiently attaching cells. The porous body here has, for example, a porosity of 30 to 95%.
However, when culturing cells with strong adhesion to the culture carrier, it is necessary to suppress adhesion, and therefore the inner surface of the groove 1 is preferably a dense body. The dense body here has, for example, a porosity of 95% or more.
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］
直径１３ｍｍのハイドロキシアパタイトの円板状の基材表面に、平行ライン状に、幅１８０μｍ、深さ４０μｍの溝を、５０μｍ間隔で形成した図１に示すような細胞培養担体を作製した。この細胞培養担体を２４ウェルプレートの穴に入れ、Ｈｅｐ−Ｇ２（ヒト肝ガン細胞）を１．０×１０4個撒種し、１０％ＦＢＳ（fetal bovine serum；ウシ胎児血清）を混合したＤＭＥＭを用いて、５％ＣＯ2インキュベータ内で、３７℃で培養した。
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]
A cell culture carrier as shown in FIG. 1 was prepared, in which grooves having a width of 180 μm and a depth of 40 μm were formed at intervals of 50 μm on the surface of a disk-shaped substrate of hydroxyapatite having a diameter of 13 mm in parallel lines. DMEM mixed with 10% FBS (fetal bovine serum) was prepared by placing this cell culture carrier in a well of a 24-well plate, seeding 1.0 × 10 4 Hep-G2 (human hepatoma cells). And cultured at 37 ° C. in a 5% CO 2 incubator.
７日間経過後、培養担体上で増殖したＨｅｐ−Ｇ２を、トリプシン処理により剥離し、血球計算板により、細胞数を計測したところ、５．０×１０5個であった。
また、増殖した細胞を、グルタルアルデヒドで固定処理し、ＳＥＭ観察したところ、ライン状に配列している凹部内で、Ｈｅｐ−Ｇ２が細胞凝集体を形成していることが認められた。
図７−１および図７−２に、このＨｅｐ−Ｇ２の顕微鏡写真を示す。図７−１は倍率４００倍、図７−２は倍率２５００倍である。
さらに、上記において増殖したＨｅｐ−Ｇ２について、免疫測定法により、細胞１．０×１０6個当たりのアルブミン量を算出したところ、４００μｇ／日であり、ゼラチンコートディッシュにおいて増殖したＨｅｐ−Ｇ２と比較して、約６倍の物質生産能力を有していることが認められた。
After 7 days, Hep-G2 grown on the culture carrier was peeled off by trypsin treatment, and the number of cells was measured with a hemocytometer. As a result, it was 5.0 × 10 5 .
In addition, when the grown cells were fixed with glutaraldehyde and observed with SEM, it was found that Hep-G2 formed cell aggregates in the recesses arranged in a line.
FIG. 7-1 and FIG. 7-2 show micrographs of this Hep-G2. 7-1 is 400 times magnification, and FIG. 7-2 is 2500 times magnification.
Further, the amount of albumin per 1.0 × 10 6 cells of Hep-G2 grown in the above was calculated by immunoassay, which was 400 μg / day, compared with Hep-G2 grown in gelatin-coated dishes. Thus, it was found that the substance production capacity was about 6 times.
［比較例１］
直径１３ｍｍのハイドロキシアパタイトの平らな円板状の細胞培養担体を用いて、実施例１と同様にして、Ｈｅｐ−Ｇ２（ヒト肝ガン細胞）を培養した。
７日間経過後、培養担体上で増殖したＨｅｐ−Ｇ２を、トリプシン処理により剥離し、血球計算板により、細胞数を計測したところ、１．０×１０5個であった。
また、平板上で増殖した細胞を、グルタルアルデヒドで固定処理し、ＳＥＭ観察したところ、凝集体を形成せずに、扁平状に増殖していることが認められた。
図８−１および図８−２に、このＨｅｐ−Ｇ２の顕微鏡写真を示す。図８−１は倍率５００倍、図８−２は倍率２５００倍である。
さらに、上記において増殖したＨｅｐ−Ｇ２について、免疫測定法により、細胞１．０×１０6個当たりのアルブミン量を算出したところ、４０μｇ／日であり、ゼラチンコートディッシュにおいて増殖したＨｅｐ−Ｇ２と比較して、物質生産能力は同等程度であることが認められた。
[Comparative Example 1]
Hep-G2 (human hepatoma cells) was cultured in the same manner as in Example 1 using a flat disk-shaped cell culture carrier of hydroxyapatite having a diameter of 13 mm.
After 7 days, Hep-G2 grown on the culture carrier was peeled off by trypsin treatment, and the number of cells was measured with a hemocytometer. As a result, it was 1.0 × 10 5 .
Further, when the cells grown on the flat plate were fixed with glutaraldehyde and observed with an SEM, it was found that the cells grew flat without forming aggregates.
FIG. 8A and FIG. 8B show micrographs of this Hep-G2. 8-1 is 500 times magnification, and FIG. 8-2 is 2500 times magnification.
Further, the amount of albumin per 1.0 × 10 6 cells of Hep-G2 grown in the above was calculated by immunoassay, which was 40 μg / day, compared with Hep-G2 grown in gelatin-coated dishes. Thus, the material production capacity was found to be comparable.
１ 溝
２ 基板表面（凸部）
３ 凹部
1 groove 2 substrate surface (convex part)
3 recess
Claims (6)
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