JP2010136706A - Cell culture carrier - Google Patents
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- JP2010136706A JP2010136706A JP2008318707A JP2008318707A JP2010136706A JP 2010136706 A JP2010136706 A JP 2010136706A JP 2008318707 A JP2008318707 A JP 2008318707A JP 2008318707 A JP2008318707 A JP 2008318707A JP 2010136706 A JP2010136706 A JP 2010136706A
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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
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/12—Well or multiwell plates
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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
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/20—Material Coatings
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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
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/22—Transparent or translucent parts
Abstract
Description
本発明は、透過観察型顕微鏡による観察を可能にし、且つ基材表面にマトリクス状に配列された凹部の内表面に、選択的に細胞が接着・増殖可能な細胞培養担体に関する。 The present invention relates to a cell culture carrier that enables observation with a transmission observation microscope and that allows cells to selectively adhere to and grow on the inner surface of recesses arranged in a matrix on the surface of a substrate.
近年、細胞培養技術の向上に伴い、細胞が産生するポリアミノ酸や蛋白質類を用いた治療、さらには、細胞自身を患部に用いる先端医療などが行われるようになり、状態の良い多様な細胞が大量に必要とされるようになってきた。このため、様々な種類の細胞をｉｎ ｖｉｔｒｏ（生体外）で効率的に培養する方法が検討されている。
しかし、これらの研究の多くは、コラーゲンなどに代表される生体高分子をコーティングした２次元的なシャーレやボトル上で、様々な添加物を培地に添加して培養する方法が主流である。
In recent years, with the improvement of cell culture technology, treatment using polyamino acids and proteins produced by cells, as well as advanced medical treatment using cells themselves as affected areas, etc. have been carried out, A large amount is needed. For this reason, methods for efficiently culturing various types of cells in vitro (in vitro) have been studied.
However, most of these studies are mainly performed by adding various additives to the medium and culturing on a two-dimensional petri dish or bottle coated with a biopolymer typified by collagen.
一方、３次元構造を持たせたバイオマテリアル上で細胞培養を行うと、細胞の生存率や物質生産能が向上する傾向があることが証明されてきており、３次元培養法が注目を集めている。これらの現状から、よりｉｎ ｖｉｖｏ（生体内）環境に酷似した３次元的バイオマテリアルの開発が重要になってくると考えられる。 On the other hand, it has been proven that cell culture on biomaterials with a three-dimensional structure tends to improve cell viability and substance production ability. Yes. From these current situations, it is considered that the development of a three-dimensional biomaterial more closely resembling an in vivo environment is important.
このように、細胞を３次元的に培養するための基材として、セラミックス製の多孔体やセラミックス表面に複数の凹部（以下、マイクロポアともいう）を配列させた細胞培養担体が開発されている。
しかしながら、細胞培養担体の素材としてセラミックスを用いると、可視光を透過させることができないため、生きた状態の細胞を、培養しながら観察することができない欠点があった。
また、細胞を生きた状態で観察するためには、合成高分子や生体高分子などを用いれば、透明な培養担体が得られるが、合成高分子は細胞を接着させることができず、細胞培養担体として利用できないという問題がある。
一方、細胞を接着させるために、コラーゲン、ラミニンなどに代表される生体高分子を合成高分子の基材にコーティングすると、細胞を強力に接着させることができるがマイクロポア内外に細胞が接着してしまうので、マイクロポア内に選択的に細胞を接着させて、増殖させることができない。
Thus, as a substrate for culturing cells three-dimensionally, a ceramic porous body and a cell culture carrier in which a plurality of recesses (hereinafter also referred to as micropores) are arranged on a ceramic surface have been developed. .
However, when ceramics are used as a material for the cell culture carrier, visible light cannot be transmitted, and thus there is a drawback that living cells cannot be observed while being cultured.
In addition, in order to observe cells in a living state, if a synthetic polymer or a biopolymer is used, a transparent culture carrier can be obtained, but the synthetic polymer cannot adhere cells, and cell culture. There is a problem that it cannot be used as a carrier.
On the other hand, when biopolymers typified by collagen and laminin are coated on a synthetic polymer substrate to adhere cells, cells can be strongly adhered, but cells adhere inside and outside the micropore. As a result, the cells cannot be selectively adhered to the micropores for growth.
一方、特許文献１には、細胞培養担体を構成する少なくとも１以上の層に比重調整物質を含めることによって、細胞培養中に細胞培養担体が浮くことがなく、かつ細胞を良好に培養することが可能な細胞培養担体が開示されている。
しかしながら、特許文献１に記載されている技術は、細胞を細胞培養担体表面に接着させることを前提とするものであり、マイクロポア内に保持する点について開示されているものではなかった。 However, the technique described in Patent Document 1 is based on the premise that cells are adhered to the surface of a cell culture carrier, and has not been disclosed in terms of holding in a micropore.
本発明は、上記技術的課題によりなされたものであり、基材表面にマトリクス状に配列した凹部内に細胞を選択的に接着・増殖させ、且つ増殖の様子を透過観察型顕微鏡によって基材側から細胞を観察す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 due to the above technical problem, and selectively adheres and proliferates the cells in the recesses arranged in a matrix on the surface of the base material, and the state of the growth is observed on the base material side by a transmission observation type microscope. It is an object of the present invention to provide a cell culture carrier capable of observing cells.
本発明の一実施形態によると、基材の主成分が透明材料で形成され、その表面に細胞を培養する複数の凹部を有する細胞培養担体であって、前記複数の凹部の少なくとも内表面にはセラミックス粒子が露出されており、前記基材側から前記凹部内を観察可能であることを特徴とする細胞培養担体が提供される。 According to one embodiment of the present invention, a cell culture carrier is formed of a transparent material as a main component of a substrate and has a plurality of recesses for culturing cells on the surface thereof, and at least an inner surface of the plurality of recesses There is provided a cell culture carrier characterized in that ceramic particles are exposed and the inside of the recess can be observed from the substrate side.
前記基材には、基材との重量比で０．５％以上１．０％以下の前記セラミックス粒子が含まれていることが好ましい。 The base material preferably contains the ceramic particles in a weight ratio of 0.5% to 1.0% by weight with the base material.
前記複数の凹部の各々の開口径が１μｍ以上１０００μｍ以下であり、深さが１μｍ以上１０００μｍ以下であることが好ましい。 The opening diameter of each of the plurality of recesses is preferably 1 μm or more and 1000 μm or less, and the depth is preferably 1 μm or more and 1000 μm or less.
透明材料が、ポリ乳酸、ポリグリコール酸、アクリル樹脂、ポリエチレン、ポリスチレン、ポリプロピレン、アガロース、アクリルアミドなどの高分子のうち、少なくとも１種類以上の高分子からなることが好ましい。 The transparent material is preferably made of at least one polymer among polymers such as polylactic acid, polyglycolic acid, acrylic resin, polyethylene, polystyrene, polypropylene, agarose and acrylamide.
セラミックス粒子が、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、イットリア、シリカのうち少なくとも１種類以上のセラミックスまたはガラスからなることが好ましい。 The ceramic particles are preferably made of at least one ceramic or glass of hydroxyapatite, β-tricalcium phosphate, alumina, zirconia, titania, yttria, and silica.
本発明によれば、基材表面にマトリクス状に配列した凹部内に細胞を選択的に接着・増殖させ、且つ増殖の様子を透過観察型顕微鏡によって基材側から細胞を観察することができる細胞培養担体が提供される。 According to the present invention, cells that can selectively adhere and proliferate in the recesses arranged in a matrix on the surface of the base material, and can observe the cells from the base material side with a transmission observation type microscope. A culture carrier is provided.
以下、本発明の実施形態を、図面を参照しつつ、説明する。なお、実施形態において、同一構成要素には同一符号を付け、実施形態の間において重複する説明は省略する。 Embodiments of the present invention will be described below with reference to the drawings. Note that, in the embodiments, the same components are denoted by the same reference numerals, and redundant description among the embodiments is omitted.
図１は、本発明の第１の実施形態に係る細胞培養担体１の構成を示す平面図である。図２は、図１に示す細胞培養担体１のＡ−Ａ線矢視断面を示す図である。 FIG. 1 is a plan view showing the configuration of the cell culture carrier 1 according to the first embodiment of the present invention. FIG. 2 is a view showing a cross section taken along line AA of the cell culture carrier 1 shown in FIG.
図１に示すように、本発明の第１の実施形態に係る細胞培養担体１は主成分が透明材料で形成された基材２から構成される。この基材２にはマトリクス状に配列された複数の凹部（マイクロウェル部）３が形成されている。この複数の凹部（マイクロウェル部）３内で細胞を培養する。 As shown in FIG. 1, the cell culture carrier 1 according to the first embodiment of the present invention is composed of a base material 2 whose main component is formed of a transparent material. A plurality of concave portions (microwell portions) 3 arranged in a matrix are formed on the base material 2. Cells are cultured in the plurality of recesses (microwell portions) 3.
図２に示すように、本発明の第１の実施形態に係る細胞培養担体１においては、複数の凹部（マイクロウェル部）３の各底面７の内表面にはセラミックス粒子４が露出している。一方で、細胞培養担体１の表面のうち複数の凹部３外の領域６の表面にはセラミックス粒子が露出していない。 As shown in FIG. 2, in the cell culture carrier 1 according to the first embodiment of the present invention, the ceramic particles 4 are exposed on the inner surface of each bottom surface 7 of the plurality of recesses (microwell portions) 3. . On the other hand, ceramic particles are not exposed on the surface of the region 6 outside the plurality of recesses 3 in the surface of the cell culture carrier 1.
このように、細胞培養担体１の凹部３の底面７の内表面のみにセラミックス粒子４が露出しているために、凹部３内に細胞を保持することができる。 Thus, since the ceramic particles 4 are exposed only on the inner surface of the bottom surface 7 of the recess 3 of the cell culture carrier 1, the cells can be held in the recess 3.
本発明の第１の実施形態に係る細胞培養担体１は基材２の主成分が透明材料からなる。基材（透明材料）２の凹部３で選択的に細胞を接着・増殖し、かつ、細胞を観察するため基材２には、基材２との重量比で０．５％以上１．０％以下のセラミックス粒子４を含んでいることが好ましい。ここで、基材２に、セラミックス粒子４が０．５％未満である場合には、凹部３内で細胞を保持することができない。また、一方で、基材２に、セラミックス粒子４が１．０％を超えると、細胞培養担体１の裏面から増殖した細胞を透過観察型顕微鏡等で観察することができない。 In the cell culture carrier 1 according to the first embodiment of the present invention, the main component of the substrate 2 is made of a transparent material. In order to adhere and proliferate cells selectively in the recesses 3 of the base material (transparent material) 2 and observe the cells, the base material 2 has a weight ratio of 0.5% to 1.0% with respect to the base material 2. % Of ceramic particles 4 are preferably included. Here, when the ceramic particle 4 is less than 0.5% on the base material 2, the cells cannot be held in the recess 3. On the other hand, if the ceramic particles 4 exceed 1.0% on the substrate 2, the cells grown from the back surface of the cell culture carrier 1 cannot be observed with a transmission observation microscope or the like.
本発明の第１の実施形態に係る細胞培養担体１を構成する基材２の主成分が透明材料として、ポリ乳酸、ポリグリコール酸、アクリル樹脂、ポリエチレン、ポリスチレン、ポリプロピレン、アガロース、アクリルアミドなどの合成高分子やアガロースに代表される糖類のうち、少なくとも１種類以上の高分子からなることが好ましい。これらの透明材料については、細胞毒性を示さない限り、どのようなものを用いても作製可能であるが、特に生体内に移植する目的がある場合、生分解性を示す、ポリ乳酸、グリコール酸などを用いることが好ましい。 Synthesis of polylactic acid, polyglycolic acid, acrylic resin, polyethylene, polystyrene, polypropylene, agarose, acrylamide, etc. as the main component of the substrate 2 constituting the cell culture carrier 1 according to the first embodiment of the present invention is a transparent material. Of the saccharides typified by a polymer or agarose, it is preferably composed of at least one polymer. These transparent materials can be produced using any material as long as they do not exhibit cytotoxicity. However, particularly when there is a purpose of transplanting in vivo, polylactic acid and glycolic acid exhibiting biodegradability. Etc. are preferably used.
本発明の第１の実施形態に係る基材２中に含有するセラミックス粒子４として、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、イットリア、シリカのうち少なくとも１種類以上のセラミックスまたはガラスからなることが好ましい。これらのセラミックスまたはガラスは生体安定性と細胞接着性とが確認されているため、好適に用いることができる。 As ceramic particles 4 contained in the substrate 2 according to the first embodiment of the present invention, at least one kind of ceramics selected from hydroxyapatite, β-tricalcium phosphate, alumina, zirconia, titania, yttria, and silica or It is preferably made of glass. Since these ceramics or glass has been confirmed to be biostable and cell-adhesive, they can be suitably used.
本発明の第１の実施形態に係る凹部３の各々の開口径は１μｍ以上１０００μｍ以下であり、深さが１μｍ以上１０００μｍ以下であることが好ましい。通常、細胞のサイズは１０μｍ以上２０μｍ以下であることから、凹部３で細胞が選択的に保持・増殖することを考慮すると、前記開口径・深さともに５０μｍ以上１０００μｍ以下であることがさらに好ましい。 The opening diameter of each recess 3 according to the first embodiment of the present invention is preferably 1 μm or more and 1000 μm or less, and the depth is preferably 1 μm or more and 1000 μm or less. Usually, since the size of the cell is 10 μm or more and 20 μm or less, it is more preferable that both the opening diameter and the depth are 50 μm or more and 1000 μm or less in consideration of the selective retention and proliferation of the cells in the recess 3.
本発明の第１の実施形態に係る細胞培養担体１の凹部３は、マトリクス状に規則的に配列していることが好ましい。さらに、凹部３間の距離が１０μｍ以上５０μｍ以下であることがより好ましい。こうすることで、細胞を撒種した際、細胞培養担体１の凹部３に、細胞をより効率的に接着させ、且つ十分に細胞に栄養分・酸素を供給することができる。ここで、マトリクス状とは、行方向及び列方向に配列されていることを意味し、特に、行方向と列方向とが、直交していることが好ましい。このような形状の細胞培養担体１を３次元構造の細胞培養担体１として用いることにより、凹部３内で細胞を確実に保持させて、凹部３内で安定して細胞を培養することができる。 It is preferable that the recesses 3 of the cell culture carrier 1 according to the first embodiment of the present invention are regularly arranged in a matrix. Furthermore, the distance between the recesses 3 is more preferably 10 μm or more and 50 μm or less. By so doing, when the cells are seeded, the cells can be more efficiently adhered to the recesses 3 of the cell culture carrier 1, and nutrients and oxygen can be sufficiently supplied to the cells. Here, the matrix shape means that the matrix is arranged in the row direction and the column direction, and it is particularly preferable that the row direction and the column direction are orthogonal to each other. By using the cell culture carrier 1 having such a shape as the cell culture carrier 1 having a three-dimensional structure, the cells can be reliably held in the recesses 3 and the cells can be stably cultured in the recesses 3.
また、本発明の第１の実施形態に係る細胞の培養方法は、上記のような本発明の実施形態に係る細胞培養担体１を用いて、未分化細胞を細胞培養担体１の凹部３の少なくとも１ヶ所に撒種して培養し、細胞塊を得ることを特徴とするものである。上記のような特定の形状を有する細胞培養担体１を用いることにより、効率的に細胞の３次元細胞塊を形成させることができ、且つ細胞塊を細胞培養担体１から容易に透過観察型顕微鏡で観察することができる。 In addition, the cell culture method according to the first embodiment of the present invention uses the cell culture carrier 1 according to the embodiment of the present invention as described above to remove undifferentiated cells at least in the recesses 3 of the cell culture carrier 1. It is characterized by seeding and culturing at one place to obtain a cell mass. By using the cell culture carrier 1 having the specific shape as described above, a three-dimensional cell mass of cells can be efficiently formed, and the cell mass can be easily removed from the cell culture carrier 1 with a transmission observation type microscope. Can be observed.
本発明の第１の実施形態に係る細胞培養担体１において培養する細胞は、接着系細胞である。具体的には、ＥＳ細胞、ｉＰＳ細胞、間葉系幹細胞に代表される未分化細胞や、肝臓や腎臓由来の正常組織細胞などが挙げられる。なお、ここでいう未分化細胞とは、自分自身と同じ細胞に増殖する能力と、分化誘導因子の付与により決まった組織細胞に増殖する能力と、分化誘導因子の付与により決まった組織細胞に分化する能力とを有している、未分化の状態にある細胞であり、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細胞のことをいう。 The cells cultured in the cell culture carrier 1 according to the first embodiment of the present invention are adherent cells. Specific examples include undifferentiated cells such as ES cells, iPS cells, and mesenchymal stem cells, and normal tissue cells derived from the liver and kidney. The undifferentiated cells mentioned here are the ability to proliferate to the same cells as themselves, the ability to proliferate to tissue cells determined by the application of differentiation-inducing factors, and the differentiation into tissue cells determined by the application of differentiation-inducing factors. It is a cell in an undifferentiated state having the ability to perform, and once it has differentiated into another tissue cell, it cannot return to the undifferentiated state.
本発明は、以上のとおり構成することによって、培養した細胞を非常に簡単に裏面側から観察することができる。さらに、細胞培養担体１の凹部３外の領域６には、セラミックス粒子４が含まれていないため、細胞が浮いた状態となり、前記領域６上に細胞が接地された場合でも、容易に凹部３内に細胞を入れることができる。 By configuring the present invention as described above, the cultured cells can be observed very easily from the back side. Further, since the region 6 outside the recess 3 of the cell culture carrier 1 does not contain the ceramic particles 4, the cells are in a floating state, and even when the cells are grounded on the region 6, the recess 3 can be easily formed. You can put cells inside.
次に、図３を用いて、本発明の第２の実施形態を示す。これは凹部３の底面７の内表面のみならず側面８の内表面にセラミックス粒子４を露出させた例である。 Next, a second embodiment of the present invention will be described with reference to FIG. This is an example in which the ceramic particles 4 are exposed not only on the inner surface of the bottom surface 7 of the recess 3 but also on the inner surface of the side surface 8.
本発明の第２の実施形態においても、その上面からみた構成は図１と同様である。また、基材２の材料、セラミックス粒子４の材料、基材２中の含有率、凹部３の形状、表面の深さ等も本発明の第１の実施形態において説明したものと同様である。 Also in the second embodiment of the present invention, the configuration viewed from the top is the same as in FIG. Moreover, the material of the base material 2, the material of the ceramic particles 4, the content in the base material 2, the shape of the recess 3, the depth of the surface, and the like are the same as those described in the first embodiment of the present invention.
本発明の第２の実施形態の細胞培養担体１は凹部３の底面７と側面８との各内表面にセラミックス粒子４を露出しているために、凹部３内に効果的に細胞を保持することができ、かつ、培養した細胞を側面及び裏面側から観察することができる。さらに、細胞培養担体１の凹部３外の領域６には、セラミックス粒子４が形成されていないため、細胞が浮いた状態となり、容易に凹部３内に細胞を入れることができる。 Since the cell culture carrier 1 of the second embodiment of the present invention exposes the ceramic particles 4 on the inner surfaces of the bottom surface 7 and the side surface 8 of the recess 3, the cells are effectively retained in the recess 3. And the cultured cells can be observed from the side and the back side. Further, since the ceramic particles 4 are not formed in the region 6 outside the recess 3 of the cell culture carrier 1, the cells are in a floating state, and the cells can be easily put into the recess 3.
以下、本発明の各実施形態におけるセラミックス粒子の含有量を決定するにあたってなした実験結果を示す。 Hereafter, the experimental result made in determining content of the ceramic particle in each embodiment of this invention is shown.
アガロース１００ｍｇと純水４９００ｍｇとを混合し、ここに、５０％ハイドロキシアパタイトスラリーを０ｍｇ、２５ｍｇ、５０ｍｇ、１００ｍｇと振って添加し、各々超音波処理を５分間行った。超音波処理後、混合液を１００℃で５分間加熱した後、鋳型に流し込み、室温で３０分静置した。３０分静置後、鋳型より離型した細胞培養担体を透過観察型顕微鏡により観察した。 100 mg of agarose and 4900 mg of pure water were mixed, and 50% hydroxyapatite slurry was shaken and added thereto at 0 mg, 25 mg, 50 mg, and 100 mg, and sonication was performed for 5 minutes. After sonication, the mixture was heated at 100 ° C. for 5 minutes, then poured into a mold and allowed to stand at room temperature for 30 minutes. After standing for 30 minutes, the cell culture carrier released from the template was observed with a transmission observation microscope.
５０％ハイドロキシアパタイトスラリーを５０ｍｇ添加した時の、細胞培養担体１の透過観察型顕微鏡写真（１００倍）を図４に示す。上記４条件いずれも透過性を有することが確認された。 FIG. 4 shows a transmission observation type micrograph (100 times) of the cell culture carrier 1 when 50 mg of 50% hydroxyapatite slurry is added. It was confirmed that all of the above four conditions have permeability.
さらに、２４ウェルプレート中に、作製したそれぞれの細胞培養担体１を浸漬させ、あらかじめ培養したＥＳ細胞を５．０×１０５個、撤種し、ＫＳＲ（Ｋｎｏｃｋｏｕｔ Ｓｃｒａｍ Ｒｅｐｌａｃｃｍｅｎｔ）、ピルビン酸、非必須アミノ酸、ＬＩＦ（Ｌｅｕｋｅｍｉａ Ｉｎｈｉｂｉｔｏｒｙ Ｆａｃｔｏｒ）を含んだＤＭＥＭにおいて、５％ＣＯ２インキュベーターを用い、３７℃で３日間、培養した。 Furthermore, each prepared cell culture carrier 1 is immersed in a 24-well plate, 5.0 × 10 5 ES cells cultured in advance are removed, KSR (Knockout Scram Replacement), pyruvate, non-essential In DMEM containing the amino acid LIF (Leukemia Inhibitory Factor), the cells were cultured at 37 ° C. for 3 days using a 5% CO 2 incubator.
培養後の細胞培養担体１を透過観察型顕微鏡で観察したところ、５０％ハイドロキシアパタイトスラリーを０ｍｇ、２５ｍｇ添加した時の細胞培養担体上には、ほとんどの細胞が接着せず、細胞の培養が確認できなかった。一方、５０％ハイドロキシアパタイトスラリーを５０ｍｇ、１００ｍｇ添加した細胞培養担体１上の凹部には細胞が接着・増殖する様子を確認できた。 When the cell culture carrier 1 after culturing was observed with a transmission observation type microscope, most cells did not adhere to the cell culture carrier when 0 mg and 25 mg of 50% hydroxyapatite slurry were added, and cell culture was confirmed. could not. On the other hand, it was confirmed that cells were adhered and proliferated in the recesses on the cell culture carrier 1 to which 50 mg and 100 mg of 50% hydroxyapatite slurry were added.
アガロース１００ｍｇと純水４９００ｍｇとを混合し、さらに、５０％ハイドロキシアパタイトスラリーを５００ｍｇ添加し、超音波処理を５分間行った。超音波処理後、混合液を１００℃で５分間加熱した後、鋳型に流し込み、室温で３０分静置した。３０分静置後、鋳型より離型した。 100 mg of agarose and 4900 mg of pure water were mixed, 500 mg of 50% hydroxyapatite slurry was further added, and sonication was performed for 5 minutes. After sonication, the mixture was heated at 100 ° C. for 5 minutes, then poured into a mold and allowed to stand at room temperature for 30 minutes. After leaving still for 30 minutes, it was released from the mold.
さらに、２４ウェルプレート中に、作製した細胞培養担体１を浸漬させ、あらかじめ培養したＥＳ細胞を５．０×１０５個、撤種し、ＫＳＲ（Ｋｎｏｃｋｏｕｔ Ｓｃｒａｍ Ｒｅｐｌａｃｃｍｅｎｔ）、ピルビン酸、非必須アミノ酸、ＬＩＦ（Ｌｅｕｋｅｍｉａ Ｉｎｈｉｂｉｔｏｒｙ Ｆａｃｔｏｒ）を含んだＤＭＥＭにおいて、５％ＣＯ２インキュベーターを用い、３７℃で３日間、培養した。 Further, the prepared cell culture carrier 1 is immersed in a 24-well plate, 5.0 × 10 5 ES cells cultured in advance are removed, KSR (Knockout Scram Replacement), pyruvate, non-essential amino acids, In DMEM containing LIF (Leukemia Inhibitory Factor), the cells were cultured at 37 ° C. for 3 days using a 5% CO 2 incubator.
培養後の細胞培養担体１を透過観察型顕微鏡で観察したが、可視光が透過せず、細胞が接着・増殖したことを確認できなかった。 The cultured cell culture carrier 1 was observed with a transmission observation microscope, but no visible light was transmitted, and it was not possible to confirm that the cells adhered and proliferated.
上記各実験から、５０％ハイドロキシアパタイトスラリーを５０ｍｇ、１００ｍｇ添加した細胞培養担体１が細胞の付着及び透過性の観点から最適であることが判明した。これは、基材との重量比で０．５％以上１．０％以下の量のセラミックス粒子４を含ませることに相当する。 From the above experiments, it was found that the cell culture carrier 1 to which 50 mg and 100 mg of 50% hydroxyapatite slurry were added was optimal from the viewpoint of cell adhesion and permeability. This corresponds to including the ceramic particles 4 in an amount of 0.5% to 1.0% by weight ratio to the base material.
１…細胞培養担体、２…基材、３…凹部（マイクロウェル部）、４…セラミックス粒子、５…凹部間の領域、７…底面、８…側面 DESCRIPTION OF SYMBOLS 1 ... Cell culture carrier, 2 ... Base material, 3 ... Recessed part (microwell part), 4 ... Ceramic particle, 5 ... Area between recessed parts, 7 ... Bottom face, 8 ... Side face
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