JP4660713B2 - Cell adhesion material - Google Patents
Cell adhesion material Download PDFInfo
- Publication number
- JP4660713B2 JP4660713B2 JP2003274530A JP2003274530A JP4660713B2 JP 4660713 B2 JP4660713 B2 JP 4660713B2 JP 2003274530 A JP2003274530 A JP 2003274530A JP 2003274530 A JP2003274530 A JP 2003274530A JP 4660713 B2 JP4660713 B2 JP 4660713B2
- Authority
- JP
- Japan
- Prior art keywords
- atomic oxygen
- irradiation
- fep
- cell
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/582—Recycling of unreacted starting or intermediate materials
Landscapes
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Treatments Of Macromolecular Shaped Articles (AREA)
Description
本発明は、細胞培養に用いられる各種医療用材料として使用可能な細胞接着材料に関するものである。 The present invention relates to a cell adhesion material that can be used as various medical materials used for cell culture.
生体と人工材料との適合メカニズムの理解が深まるにつれ、新規生体材料の研究開発やその実用化が、様々な分野で進められてきた。特に、インプラントや人工血管などに用いられる生体材料においては、材料表面が基本的な生体機能の発現に密接に関連していることから、生体の最小単位である細胞と材料表面との親和性の向上が求められている。 As the understanding of the mechanism of adaptation between living organisms and artificial materials has deepened, research and development of new biomaterials and their practical application have been promoted in various fields. In particular, in the biomaterials used for implants and artificial blood vessels, the surface of the material is closely related to the expression of basic biological functions. There is a need for improvement.
これまでにも、材料表面と細胞との親和性に関する様々な研究が行われてきた（非特許文献１−３）。例えば、細胞接着阻害タンパク質や細胞接着タンパク質などを材料表面に修飾し、細胞接着を制御することで、材料表面において細胞接着パターンを作成した研究が報告されている（非特許文献４，５）。これらの研究の多くは、基板となる材料表面へのタンパク質等の修飾によるものであり、様々な細胞機能の制御が期待される。しかしながら、複雑な表面処理が必要であり、また、生体内では、修飾したタンパク質自体が、様々な生化学的反応を受けるため、安定した接着制御の維持が容易ではない。そのため、基板材料表面自体の組成や構造の直接的な改質による細胞親和性を向上させる方法も提案されている（特許文献１）。
しかしながら、細胞接着阻害タンパク質や細胞接着タンパク質などを材料表面に修飾し、細胞接着を制御する方法の場合、基板と細胞間の接着力が比較的弱いという問題がある。また、これらタンパク質により材料表面を修飾した場合、基板材料の再利用を行うことが困難であるという問題もある。さらに、特許文献１のものは、イオンを注入することによって基板表面の化学構造を変化させ、基板表面の化学的特性を変化させたものである。また、プラズマ等によって基板表面を物理的に改質する方法も知られているが、この特許文献１にも記載されているように、細胞の付着を促進する反面、増殖を抑制する傾向にある。また、プラズマの状態は、装置ごとに異なる為、処理条件の統一化が困難であり、実用化することが困難である。 However, in the case of a method for controlling cell adhesion by modifying cell adhesion inhibiting protein or cell adhesion protein on the surface of the material, there is a problem that the adhesive force between the substrate and the cells is relatively weak. Further, when the material surface is modified with these proteins, there is a problem that it is difficult to reuse the substrate material. Furthermore, the thing of patent document 1 changes the chemical structure of the substrate surface by injecting ion, and changes the chemical characteristic of the substrate surface. In addition, a method of physically modifying the substrate surface with plasma or the like is also known, but as described in Patent Document 1, it promotes cell adhesion but tends to suppress proliferation. . In addition, since the plasma state differs depending on the apparatus, it is difficult to unify the processing conditions and it is difficult to put it into practical use.
そこで本発明は、基板材料の表面に原子状ビームを照射することにより、表面を物理的に変化させ、これにより、基板材料表面と細胞との親和性を制御することで、細胞との親和性を向上させた細胞接着材料を提供することを目的とする。 Therefore, the present invention changes the surface physically by irradiating the surface of the substrate material with an atomic beam, thereby controlling the affinity between the surface of the substrate material and the cell, thereby improving the affinity with the cell. An object of the present invention is to provide a cell adhesion material with improved resistance.
本発明は、前述の課題を解決する為になされたものであり、すなわち、本発明に係る細胞接着材料は、レーザーデトネーション現象により、酸素分子を解離、加速して得られる原子状酸素ビームを、ＦＥＰ（４フッ化エチレン・６フッ化プロピレン共重合樹脂）からなる基板材料の表面に照射することにより、前記基板材料表面が物理的に改質されてなるものである。また、前記原子状酸素ビームの照射量が、１．５×１０ 1９ ａｔｏｍｓ／ｃｍ2以上であるものである。 The present invention has been made in order to solve the above-mentioned problems, that is, the cell adhesion material according to the present invention uses an atomic oxygen beam obtained by dissociating and accelerating oxygen molecules by a laser detonation phenomenon. by morphism irradiation on the surface of a substrate material consisting of FEP (4 tetrafluoroethylene-hexafluoropropylene copolymer resin), the substrate material surface is made by physically modified. Also, the dose of the atomic oxygen beam is what is 1.5 × 10 19 atoms / cm 2 or more.
本発明に係る細胞接着材料は、原子状酸素ビームの照射により表面改質を行い、原子状酸素ビームの照射による表面に形成された新たな凹凸によりぬれ性が低下し、撥水性が増加するとともに、細胞親和性の向上が可能となる。 The cell adhesion material according to the present invention undergoes surface modification by irradiation with an atomic oxygen beam, and the wettability decreases due to new unevenness formed on the surface by irradiation with the atomic oxygen beam, and the water repellency increases. It is possible to improve cell affinity.
以下、図面を参照しつつ、本発明に係る細胞接着材料を実施する為の最良の形態の一例を具体的に説明する。 Hereinafter, an example of the best mode for carrying out the cell adhesion material according to the present invention will be specifically described with reference to the drawings.
本発明に係る細胞接着材料の基板材料としては、一般的な高分子材料を使用することができ、ＦＥＰ、ＬＤＰＥ（低密度ポリエチレン）等を例示することができ、特に、ＦＥＰにおいて顕著な効果を得ることができる。 As a substrate material of the cell adhesion material according to the present invention, a general polymer material can be used, and examples thereof include FEP, LDPE (low density polyethylene) and the like, and in particular, a remarkable effect in FEP. Obtainable.
そして、これら基板材料の表面を改質するためのレーザーデトネーション型原子状ビーム発生装置の概略図を図１に示す。本装置は、図１に示すように、ピエゾ駆動のパルスバルブにより真空チャンバー内に導入された酸素分子に５〜７Ｊ／Ｐｕｌｓｅの炭酸ガスレーザー光を集光し、レーザーデトネーション現象により、酸素分子を解離、加速することで、原子状酸素を、５ｅＶ程度の並進エネルギーを有するビームとして材料表面に照射することができる。生成された原子状酸素ビームのフラックスおよび並進エネルギーは、水晶振動子マイクロバランスと飛行時間スペクトルを用いて、“その場（ｉｎ−ｓｉｔｕ）”測定することができる。 A schematic diagram of a laser detonation type atomic beam generator for modifying the surface of these substrate materials is shown in FIG. As shown in FIG. 1, this apparatus focuses 5-7 J / Pulse carbon dioxide laser light on oxygen molecules introduced into the vacuum chamber by a piezo-driven pulse valve, and converts the oxygen molecules by laser detonation. By dissociating and accelerating, the surface of the material can be irradiated with atomic oxygen as a beam having a translational energy of about 5 eV. The flux and translational energy of the generated atomic oxygen beam can be measured “in-situ” using a quartz crystal microbalance and a time-of-flight spectrum.
原子状酸素の照射量は、５．０×１０18ａｔｏｍｓ／ｃｍ2以上、好ましくは１．５×１０19ａｔｏｍｓ／ｃｍ2以上であることが好ましい。 The irradiation amount of atomic oxygen is 5.0 × 10 18 atoms / cm 2 or more, preferably 1.5 × 10 19 atoms / cm 2 or more.
以下、実施例により、本発明に係る細胞接着材料を、より具体的に説明する。基板材料としては、ＦＥＰを使用し、厚さ５０μｍのシートから直径１８ｍｍの円板試料を作成した。シート状試料を使用することで、材料表面に接着した細胞の透過光による観察が可能となる。なお、ＦＥＰとの比較のために、ＬＤＰＥを基板として、ＦＥＰと同様にして厚さ５０μｍのシートから直径１８ｍｍの円板試料を作成した。 Hereinafter, the cell adhesion material according to the present invention will be described more specifically with reference to examples. As a substrate material, FEP was used, and a disk sample having a diameter of 18 mm was prepared from a sheet having a thickness of 50 μm. By using a sheet-like sample, it is possible to observe the cell adhered to the material surface with transmitted light. For comparison with FEP, a disk sample having a diameter of 18 mm was prepared from a 50 μm thick sheet using LDPE as a substrate in the same manner as FEP.
各基板となる試料は、エチルアルコール、ジエチルエーテルおよび純水で超音波洗浄を行った。洗浄後、試料を真空チャンバー内に入れ、原子ビームを照射した。なお、原子状酸素照射条件は、原子状酸素の並進エネルギー約５．０ｅＶ、原子状酸素のフラックス８．４×１０15ａｔｏｍｓ／ｃｍ2／ｓであり、原子状酸素の照射量が、１．５×１０18ａｔｏｍｓ／ｃｍ2、５．０×１０18ａｔｏｍｓ／ｃｍ2、１．５×１０19ａｔｏｍｓ／ｃｍ2、および６．０×１０19ａｔｏｍｓ／ｃｍ2の４つの改質表面を作成した。 The sample to be each substrate was subjected to ultrasonic cleaning with ethyl alcohol, diethyl ether and pure water. After cleaning, the sample was placed in a vacuum chamber and irradiated with an atomic beam. The atomic oxygen irradiation conditions are as follows: the translational energy of atomic oxygen is about 5.0 eV, the atomic oxygen flux is 8.4 × 10 15 atoms / cm 2 / s, and the atomic oxygen irradiation amount is 1. Four modified surfaces were created: 5 × 10 18 atoms / cm 2 , 5.0 × 10 18 atoms / cm 2 , 1.5 × 10 19 atoms / cm 2 , and 6.0 × 10 19 atoms / cm 2 did.
原子状酸素照射前後における基板の表面性状の評価は、ＸＰＳによる表面化学組成および化学状態分析、接触角測定によるぬれ性の評価、ならびに、ＡＦＭによる表面構造観察により行った。 The evaluation of the surface properties of the substrate before and after atomic oxygen irradiation was performed by analyzing the surface chemical composition and chemical state by XPS, evaluating the wettability by measuring the contact angle, and observing the surface structure by AFM.
原子状酸素ビーム照射による表面酸素濃度の変化をＸＰＳを用いて測定した。測定は各試料表面上の任意の２点で行い、サンプル数は、ＦＥＰおよびＬＤＰＥに対して、それぞれｎ＝３である。 Changes in the surface oxygen concentration due to atomic oxygen beam irradiation were measured using XPS. The measurement is performed at two arbitrary points on each sample surface, and the number of samples is n = 3 for FEP and LDPE, respectively.
照射した原子状酸素の照射量とＬＤＰＥ表面における酸素濃度の関係を図２に示す。ＬＤＰＥ表面においては、原子状酸素の照射量の違いにより酸素濃度に差が見られ、原子状酸素の照射量の増加と共に、酸素濃度が有意（ｐ＜０．００１、ＡＮＯＶＡ）に上昇する傾向が見られた。 FIG. 2 shows the relationship between the amount of irradiated atomic oxygen and the oxygen concentration on the LDPE surface. On the LDPE surface, there is a difference in the oxygen concentration due to the difference in the amount of atomic oxygen irradiation, and the oxygen concentration tends to increase significantly (p <0.001, ANOVA) as the amount of atomic oxygen irradiation increases. It was seen.
一方、ＦＥＰ表面においては、図３に示すように、原子状酸素の照射量によらず有意な酸素濃度の上昇は見られなかった。 On the other hand, on the FEP surface, as shown in FIG. 3, no significant increase in oxygen concentration was observed regardless of the amount of atomic oxygen irradiation.
原子状酸素ビーム照射にともなう材料表面構造の変化をＡＦＭを用いて観察した。また、観察画像から表面粗さを計測した。観察は、各試料表面上の任意の２点で行い、サンプル数は、ＦＥＰおよびＬＤＰＥに対して、それぞれｎ＝４である。観察領域の大きさは５μｍ×５μｍであり、コンタクトモードで観察した。 Changes in the material surface structure with atomic oxygen beam irradiation were observed using AFM. Further, the surface roughness was measured from the observed image. Observation is performed at two arbitrary points on the surface of each sample, and the number of samples is n = 4 for FEP and LDPE, respectively. The size of the observation area was 5 μm × 5 μm and was observed in the contact mode.
まず、原子状酸素ビーム未照射のＬＤＰＥ表面のＡＦＭ観察画像を図４（ａ）に、原子状酸素を６．０×１０19ａｔｏｍｓ／ｃｍ2照射したＬＤＰＥ表面の観察画像を図４（ｂ）に示す。また、原子状酸素の照射量とＬＤＰＥ表面の算術平均粗さ（Ｒａ）との関係を図５に示す。ＡＦＭによるＬＤＰＥ表面の観察より、ＬＤＰＥ表面では、原子状酸素ビーム照射による顕著な構造変化は認められなかった。また、表面粗さにおいても、原子状酸素ビーム照射による有意な変化は見られなかった。 First, FIG. 4A shows an AFM observation image of an LDPE surface not irradiated with an atomic oxygen beam, and FIG. 4B shows an observation image of an LDPE surface irradiated with atomic oxygen 6.0 × 10 19 atoms / cm 2 . Shown in FIG. 5 shows the relationship between the irradiation dose of atomic oxygen and the arithmetic average roughness (Ra) of the LDPE surface. From the observation of the LDPE surface by AFM, no significant structural change was observed on the LDPE surface due to the atomic oxygen beam irradiation. Also, the surface roughness was not significantly changed by the atomic oxygen beam irradiation.
次に、原子状酸素ビーム未照射のＦＥＰ表面のＡＦＭ観察画像を図６（ａ）に、原子状酸素を６．０×１０19ａｔｏｍｓ／ｃｍ2照射したＦＥＰ表面の観察画像を図６（ｂ）に示す。また、原子状酸素の照射量とＦＥＰ表面の表面粗さとの関係を図７に示す。ＡＦＭによるＦＥＰ表面の観察より、原子状酸素未照射のＦＥＰ表面は、高さ１０ｎｍ程度の凹凸構造を有していることがわかった。一方、原子状酸素ビームを照射したＦＥＰ表面では、原子状酸素未照射表面と比較して、凹凸構造の高さが大きくなっており、原子状酸素の照射量の増加と共にその凹凸構造の高さが、有意（ｐ＜０．００１、ＡＮＯＶＡ）に増加する傾向が見られた。また、原子状酸素を１．５×１０19ａｔｏｍｓ／ｃｍ2以上照射した表面では、高さ１００ｎｍ以上の新たな凹凸構造の形成が観察された。 Next, FIG. 6A shows an AFM observation image of the FEP surface not irradiated with the atomic oxygen beam, and FIG. 6B shows an observation image of the FEP surface irradiated with atomic oxygen 6.0 × 10 19 atoms / cm 2 . ). FIG. 7 shows the relationship between the amount of atomic oxygen irradiation and the surface roughness of the FEP surface. From observation of the FEP surface by AFM, it was found that the FEP surface not irradiated with atomic oxygen had a concavo-convex structure with a height of about 10 nm. On the other hand, on the FEP surface irradiated with the atomic oxygen beam, the height of the concavo-convex structure is larger than that on the surface not irradiated with atomic oxygen, and the height of the concavo-convex structure increases as the amount of atomic oxygen irradiation increases. However, a tendency to increase significantly (p <0.001, ANOVA) was observed. In addition, formation of a new concavo-convex structure with a height of 100 nm or more was observed on the surface irradiated with atomic oxygen of 1.5 × 10 19 atoms / cm 2 or more.
原子状酸素ビーム照射による材料表面の純水の接触角変化を接触角測定装置を用いて、Ｓｅｓｓｉｌｅ Ｄｒｏｐ法により測定した。Ｓｅｓｓｉｌｅ Ｄｒｏｐ法では、表面に液滴を置いた直後は、液滴が拡がるため、大気、液滴および表面の交わる三層境界は前進し、その後、液滴の蒸発によりその境界は後退する。そこで、液滴を表面に置いた直後からの接触角の経時的変化を測定し、時刻０における接触角の外挿値を前進接触角とした。また、三層境界が後退している間、接触角はほぼ一定値をとり、これを後退接触角とした。この方法により、動的接触角を精度良く測定することができる。液滴のサイズは、約２．０μｍであり、重力の影響は無視できる。測定は、各試料表面上の任意の２点で行い、サンプル数は、ＬＤＰＥおよびＦＥＰに対して、それぞれｎ＝３である。 Changes in the contact angle of pure water on the surface of the material due to irradiation with an atomic oxygen beam were measured by a Sessile Drop method using a contact angle measuring device. In the Sessile Drop method, immediately after a droplet is placed on the surface, the droplet spreads, so that the three-layer boundary where the atmosphere, the droplet and the surface intersect with each other advances, and then the boundary recedes due to evaporation of the droplet. Accordingly, the change with time of the contact angle immediately after placing the droplet on the surface was measured, and the extrapolated value of the contact angle at time 0 was defined as the forward contact angle. Further, while the boundary between the three layers was receding, the contact angle took a substantially constant value, and this was taken as the receding contact angle. By this method, the dynamic contact angle can be accurately measured. The droplet size is about 2.0 μm, and the influence of gravity is negligible. The measurement is performed at two arbitrary points on each sample surface, and the number of samples is n = 3 for LDPE and FEP, respectively.
図８（ａ）に原子状酸素未照射のＬＤＰＥ表面、図８（ｂ）に原子状酸素６．０×１０19ａｔｏｍｓ／ｃｍ2照射後のＬＤＰＥ表面における純水の液滴の写真を示す。また、図９に原子状酸素の照射量とＬＤＰＥ表面における接触角の関係を示す。原子状酸素未照射のＬＤＰＥ表面での純水の前進接触角は、約１００°であり、後退接触角は、約６０°であった。これに対して、原子状酸素６．０×１０19ａｔｏｍｓ／ｃｍ2照射後の表面では、前進接触角が約４０°まで減少し、後退接触角も約２０°まで減少した。このように、ＬＤＰＥ表面においては、原子状酸素の照射量の増加にともなって、前進および後退接触角が、共に有意（ｐ＜０．００１、ＡＮＯＶＡ）に減少する傾向が見られた。 FIG. 8A shows a photograph of pure water droplets on the LDPE surface not irradiated with atomic oxygen, and FIG. 8B shows a pure water droplet on the LDPE surface after irradiation with atomic oxygen of 6.0 × 10 19 atoms / cm 2 . FIG. 9 shows the relationship between the dose of atomic oxygen and the contact angle on the LDPE surface. The advancing contact angle of pure water on the surface of LDPE not irradiated with atomic oxygen was about 100 °, and the receding contact angle was about 60 °. In contrast, on the surface after irradiation with atomic oxygen 6.0 × 10 19 atoms / cm 2 , the advancing contact angle decreased to about 40 ° and the receding contact angle also decreased to about 20 °. Thus, on the LDPE surface, the advancing and receding contact angles tended to decrease significantly (p <0.001, ANOVA) as the amount of atomic oxygen irradiation increased.
次に、図１０（ａ）に原子状酸素未照射のＦＥＰ表面、図１０（ｂ）に原子状酸素６．０×１０19ａｔｏｍｓ／ｃｍ2照射後のＦＥＰ表面における純水の液滴の写真を示す。また、図１１に原子状酸素の照射量とＦＥＰ表面における接触角との関係を示す。原子状酸素未照射のＦＥＰ表面での純水の前進接触角は、約１１０°であり、後退接触角は、約１００°であった。原子状酸素５．０×１０18ａｔｏｍｓ／ｃｍ2までの照射では、ほとんど変化は見られなかったが、その後、原子状酸素の照射量の増加にともなって前進および後退接触角とも増加する傾向が見られた。原子状酸素６．０×１０19ａｔｏｍｓ／ｃｍ2照射後の表面では、前進接触角が、約１４０°となり、後退接触角も約１２０°まで増加した。このように、ＦＥＰ表面においては、原子状酸素の照射量が１．５×１０19ａｔｏｍｓ／ｃｍ2以上の場合に、前進および後退接触角が共に増加する傾向がみられた。 Next, FIG. 10A shows a FEP surface not irradiated with atomic oxygen, and FIG. 10B shows a photograph of pure water droplets on the FEP surface after irradiation with atomic oxygen of 6.0 × 10 19 atoms / cm 2. Indicates. FIG. 11 shows the relationship between the dose of atomic oxygen and the contact angle on the FEP surface. The advancing contact angle of pure water on the FEP surface not irradiated with atomic oxygen was about 110 °, and the receding contact angle was about 100 °. Irradiation up to 5.0 × 10 18 atoms / cm 2 of atomic oxygen showed almost no change, but then the forward and backward contact angles tended to increase with increasing atomic oxygen dose. It was seen. On the surface after irradiation with atomic oxygen 6.0 × 10 19 atoms / cm 2 , the advancing contact angle was about 140 °, and the receding contact angle was also increased to about 120 °. Thus, on the FEP surface, both the advancing and receding contact angles tended to increase when the atomic oxygen dose was 1.5 × 10 19 atoms / cm 2 or more.
原子状酸素ビームの照射によるＬＤＰＥ表面とＦＥＰ表面の改質結果を表１にまとめて示す。 Table 1 summarizes the results of modification of the LDPE surface and the FEP surface by irradiation with an atomic oxygen beam.
表１より、ＸＰＳによる表面酸素濃度の測定の結果、ＬＤＰＥ表面においては、原子状酸素ビームの照射により、酸素濃度が増加した。しかしながら、ＦＥＰ表面においては、酸素濃度の増加は見られなかった。ＬＤＰＥ表面では、酸素濃度の増加により材料表面のぬれ性が増大し、前進および後退接触角が共に減少した。同様な現象は、ポリイミド等の他の炭化水素系高分子においても観察されている。一方、ＦＥＰ表面では、酸素濃度の変化は見られなかったが、表面粗さの増加によりぬれ性が低下し、前進および後退接触角が共に増大した。以上のように、原子状酸素ビームの照射により、ＬＤＰＥ表面のぬれ性は増大し、逆にＦＥＰ表面のぬれ性は低下した。 From Table 1, as a result of the measurement of the surface oxygen concentration by XPS, the oxygen concentration increased on the LDPE surface by irradiation with the atomic oxygen beam. However, no increase in oxygen concentration was observed on the FEP surface. On the LDPE surface, the wettability of the material surface increased with increasing oxygen concentration, and both the advancing and receding contact angles decreased. Similar phenomena have been observed in other hydrocarbon polymers such as polyimide. On the other hand, on the FEP surface, the oxygen concentration did not change, but the wettability decreased due to the increase in surface roughness, and both the advancing and receding contact angles increased. As described above, the wettability of the LDPE surface increased by the irradiation of the atomic oxygen beam, and conversely, the wettability of the FEP surface decreased.
これらの結果より、原子状酸素ビームの照射により、改質結果の異なる二つの細胞接着材料を作製することができた。ＬＤＰＥ表面では、細胞と接着基板表面との親和性における酸素濃度の影響を、ＦＥＰ表面では、表面構造の影響を検討することができる。 From these results, it was possible to produce two cell adhesion materials with different modification results by irradiation with an atomic oxygen beam. On the LDPE surface, the influence of the oxygen concentration on the affinity between the cells and the adhesion substrate surface can be examined, and on the FEP surface, the influence of the surface structure can be examined.
次に、各表面を改質した基板材料の表面に培養細胞を播種し、接着した細胞数の時間変化の測定と細胞形状の観察から、細胞と改質表面との親和性の検討を行った。 Next, cultured cells were seeded on the surface of the substrate material modified on each surface, and the affinity between the cells and the modified surface was examined by measuring the time change of the number of adhered cells and observing the cell shape. .
培養細胞には、理化学研究所細胞銀行より入手した骨芽細胞様細胞ＭＣ３Ｔ３−Ｅ１を用いた。この細胞は、コラーゲン線維等のタンパク質を細胞外に産生し、これを媒介として、基板底面に接着する。この細胞を１０％ＦＢＳを加えたα−ＭＥＭを使用し、温度３７℃、湿度１００％、５％ＣＯ2−９５％Ａｉｒの環境下で培養した。観察試料は、培地を満たしたφ＝３５ｍｍのディッシュ底に表面改質した基板材料を沈め、Ｔｒｙｐｓｉｎ／ＥＤＴＡにより分散した細胞を、約１０4ｃｅｌｌｓ／ｄｉｓｈの密度となるように播種し作製した。 As the cultured cells, osteoblast-like cells MC3T3-E1 obtained from RIKEN Cell Bank were used. These cells produce proteins such as collagen fibers outside the cell and adhere to the bottom surface of the substrate through this. The cells were cultured using α-MEM supplemented with 10% FBS in an environment of temperature 37 ° C., humidity 100%, 5% CO 2 -95% Air. An observation sample was prepared by sunk a surface-modified substrate material on a φ = 35 mm dish bottom filled with a medium, and seeding cells dispersed with Trypsin / EDTA to a density of about 10 4 cells / dish.
基板表面に接着した細胞の観察を、位相差顕微鏡（ＴＥ２００、Ｎｉｋｏｎ）を用いて、細胞播種から３０分後、２時間後、１２時間後、それ以降は１２時間ごとに９６時間後まで行った。接着細胞数の計測後、計測領域の面積で除することにより、細胞密度を算出した。細胞数計測は、各ディッシュ内の任意の２領域（２．５μｍ×３．３μｍ）で行い、サンプル数は、それぞれｎ＝３である。 Observation of the cells adhered to the substrate surface was performed using a phase contrast microscope (TE200, Nikon) 30 minutes, 2 hours, 12 hours after cell seeding, and then 96 hours after every 12 hours. . After measuring the number of adherent cells, the cell density was calculated by dividing by the area of the measurement region. The number of cells is measured in any two regions (2.5 μm × 3.3 μm) in each dish, and the number of samples is n = 3, respectively.
まず、ＬＤＰＥ表面における細胞密度の時間変化を図１２に示す。計測の結果、どの時刻おいても、原子状酸素未照射のＬＤＰＥ表面と原子状酸素を照射した各改質ＬＤＰＥ表面に接着した細胞密度に有意な差は見られなかった。 First, the time change of the cell density on the LDPE surface is shown in FIG. As a result of the measurement, there was no significant difference in the density of cells adhered to the surface of LDPE not irradiated with atomic oxygen and the surface of each modified LDPE irradiated with atomic oxygen at any time.
次に、ＦＥＰ表面における細胞密度の時間変化を図１３に示す。計測の結果、ＦＥＰ表面において、播種から４８時間以降、原子状酸素の照射量の増加に対して有意（ｐ＜０．００１、ＡＮＯＶＡ）に細胞密度が増加した。細胞播種から９６時間後、原子状酸素を６．０×１０19ａｔｏｍｓ／ｃｍ2照射して改質した表面では、細胞密度が８０ｃｅｌｌｓ／ｍｍ2となり、原子状酸素未照射の基板表面に接着した細胞数（２０ｃｅｌｌｓ／ｍｍ2）の約４倍の細胞が接着していた。また、原子状酸素を１．５×１０19ａｔｏｍｓ／ｃｍ2照射して改質した表面においても、細胞密度が５４ｃｅｌｌｓ／ｍｍ2となり、原子状酸素未照射の基板表面に接着した細胞数の約３倍の細胞が接着していた。しかしながら、原子状酸素の照射量が５．０×１０18ａｔｏｍｓ／ｃｍ2以下の照射による改質表面では、どの時刻においても、原子状酸素未照射の基板表面と比較して、接着数に有意な差は見られなかった。 Next, FIG. 13 shows the time change of the cell density on the FEP surface. As a result of the measurement, on the FEP surface, the cell density increased significantly (p <0.001, ANOVA) with respect to the increase in the dose of atomic oxygen after 48 hours from seeding. 96 hours after cell seeding, on the surface modified by irradiation with atomic oxygen at 6.0 × 10 19 atoms / cm 2 , the cell density became 80 cells / mm 2 and adhered to the substrate surface not irradiated with atomic oxygen. About four times as many cells as the number of cells (20 cells / mm 2 ) were adhered. Further, even on the surface modified by irradiating atomic oxygen with 1.5 × 10 19 atoms / cm 2 , the cell density becomes 54 cells / mm 2 , which is about the number of cells adhered to the substrate surface not irradiated with atomic oxygen. Three times as many cells were attached. However, the modified surface by irradiation with an irradiation dose of atomic oxygen of 5.0 × 10 18 atoms / cm 2 or less is significant in the number of adhesions compared with the substrate surface not irradiated with atomic oxygen at any time. There was no significant difference.
細胞播種から９６時間後に、原子状酸素未照射の基板表面と原子状酸素を照射した改質表面（原子状酸素照射量：６．０×１０19ａｔｏｍｓ／ｃｍ2）に接着した細胞の接着形状の比較を行った。原子状酸素未照射の基板表面に接着した代表的な細胞形状の位相差顕微鏡画像を図１４（ａ）に、原子状酸素を６．０×１０19ａｔｏｍｓ／ｃｍ2照射して改質した表面に接着した細胞の画像を図１４（ｂ）にそれぞれ示す。原子状酸素未照射の基板のＦＥＰ表面に接着した細胞は、改質を行った表面に接着した細胞と比較して小さく、形状も本来の骨芽細胞の形状を維持できていない。これに対して、原子状酸素を照射した基板の改質表面に接着した細胞は大きく広がり、本来の形状を維持している。 96 hours after cell seeding, the adhesion shape of the cells adhered to the substrate surface not irradiated with atomic oxygen and the modified surface irradiated with atomic oxygen (atomic oxygen irradiation amount: 6.0 × 10 19 atoms / cm 2 ) A comparison was made. Fig. 14 (a) shows a typical cell-shaped phase-contrast microscope image adhered to a substrate surface not irradiated with atomic oxygen. Fig. 14 (a) shows a surface modified by irradiation with atomic oxygen at 6.0 x 10 19 atoms / cm 2. Images of the cells adhered to are shown in FIG. The cells adhered to the FEP surface of the substrate not irradiated with atomic oxygen are smaller than the cells adhered to the modified surface, and the shape cannot maintain the original osteoblast shape. In contrast, the cells adhered to the modified surface of the substrate irradiated with atomic oxygen spread greatly and maintain their original shape.
次に、観察により得られた画像をもとに、原子状酸素未照射の基板表面と原子状酸素を照射した基板の改質表面（原子状酸素照射量：６．０×１０19ａｔｏｍｓ／ｃｍ2）に接着した個々の細胞の接着面積の定量的な比較を行った。原子状酸素未照射の基板表面と改質表面に接着した単一の骨芽細胞の接着面積の比較を図１５に示す。細胞画像の取得は、試料内の任意の領域において行い、サンプル数はそれぞれｎ＝７である。細胞接着面積は、観察から得られた画像を、画像処理ソフトに取り込み、細胞の輪郭を手動で抽出して求めた。接着面積の比較から、原子状酸素未照射の基板表面に接着した細胞より改質表面に接着した細胞の方が、図１５に示すように、有意（ｐ＜０．０１、ｔ−ｔｅｓｔ）に接着面積が大きいことがわかる。 Next, based on the image obtained by observation, the surface of the substrate not irradiated with atomic oxygen and the modified surface of the substrate irradiated with atomic oxygen (atomic oxygen irradiation amount: 6.0 × 10 19 atoms / cm 2 ) Quantitative comparison of the adhesion area of individual cells adhered to was performed. FIG. 15 shows a comparison of the adhesion area of a single osteoblast adhered to the substrate surface not irradiated with atomic oxygen and the modified surface. Cell images are acquired in an arbitrary region in the sample, and the number of samples is n = 7. The cell adhesion area was obtained by taking the image obtained from the observation into image processing software and manually extracting the outline of the cell. From the comparison of the adhesion area, the cells adhered to the modified surface were significantly (p <0.01, t-test) as shown in FIG. 15 than the cells adhered to the substrate surface not irradiated with atomic oxygen. It can be seen that the adhesion area is large.
ＬＤＰＥ表面では、原子状酸素ビームの照射により表面の酸素濃度が上昇し、ぬれ性が増大したが、原子状酸素未照射の基板表面と原子状酸素を照射した基板の改質表面の接着細胞数に有意な差は見られず、細胞の接着形状も、両者の間に明確な差が見られなかった。この結果から、ＬＤＰＥの場合、基板表面の酸素量の増加は、細胞との親和性に大きく影響を与えないと考えられる。一方、ＦＥＰ表面では、原子状酸素ビームの照射により、表面に新たな構造が形成され、ぬれ性が低下した。その結果、原子状酸素未照射の基板表面と改質表面の接着細胞数に有意な差が見られ、細胞の接着形状および接着面積においても、両者の間に有意な差が見られた。この結果から、原子状酸素ビーム照射による改質により、ＦＥＰ表面と細胞との親和性が増加したと考えることができる。すなわち、ＦＥＰのように、炭素基が表面に存在する高分子材料の場合は、原子状酸素ビームの照射により、酸素がこれら炭素基と反応し、ＣＯガスやＣＯ2ガス等となって脱離し、表面に凹凸構造を形成しやすくなるものと考えられる。 On the LDPE surface, the oxygen concentration on the surface increased by the irradiation of the atomic oxygen beam and the wettability increased, but the number of adherent cells on the substrate surface not irradiated with atomic oxygen and the modified surface of the substrate irradiated with atomic oxygen No significant difference was observed, and no clear difference was observed in the cell adhesion shape between the two. From this result, in the case of LDPE, it is considered that an increase in the amount of oxygen on the substrate surface does not significantly affect the affinity with cells. On the other hand, on the FEP surface, a new structure was formed on the surface by irradiation with the atomic oxygen beam, and the wettability decreased. As a result, a significant difference was observed in the number of adherent cells between the substrate surface that had not been irradiated with atomic oxygen and the modified surface, and a significant difference was also observed between the two in terms of the shape and area of cell adhesion. From this result, it can be considered that the affinity between the FEP surface and the cells is increased by the modification by the atomic oxygen beam irradiation. That is, in the case of a polymer material having carbon groups on the surface, such as FEP, oxygen reacts with these carbon groups by irradiation with an atomic oxygen beam, and is desorbed as CO gas, CO 2 gas, or the like. It is considered that the uneven structure is easily formed on the surface.
このように、親和性向上の要因として、原子状酸素ビームの照射により、物理的に改質されたＦＥＰ表面に新たに形成された凹凸構造が、影響を及ぼしている可能性がある。表面の凹凸構造が、細胞が自らの接着のために産生したタンパク質を材料表面に固定する役割を果たし、細胞がより接着しやすい表面状態になったと考えられる。あるいは、基板表面の凹凸構造の形成により生じた超撥水性により、細胞の産生した細胞外マトリックスと基板表面との間にこの表面吸着水層の形成が抑制され、結果として、両者間の親和性が向上したことも一因として考えられる。 Thus, as a factor for improving the affinity, the uneven structure newly formed on the physically modified FEP surface by the irradiation of the atomic oxygen beam may have an influence. It is thought that the surface uneven structure played a role of fixing proteins produced by the cells for their adhesion to the surface of the material, resulting in a surface state where the cells are more likely to adhere. Alternatively, the superhydrophobicity caused by the formation of the concavo-convex structure on the substrate surface suppresses the formation of this surface adsorbed water layer between the extracellular matrix produced by the cell and the substrate surface, resulting in an affinity between the two The improvement was also considered as a factor.
ＦＥＰ表面のパターン改質を行い、改質部と未改質部の細胞の親和性の差を利用し、細胞接着パターンの作成を試みた。図１６に示す０．５ｍｍ×２ｍｍの開口部が等間隔に並んだメッシュマスクを使用した。このメッシュマスクをＦＥＰ表面に被せ、原子状酸素ビームを照射することにより、ＦＥＰ表面のパターン改質を行った。 Pattern modification of the FEP surface was performed, and an attempt was made to create a cell adhesion pattern by utilizing the difference in affinity between cells in the modified part and the unmodified part. A mesh mask in which openings of 0.5 mm × 2 mm shown in FIG. 16 are arranged at equal intervals was used. This mesh mask was put on the FEP surface, and the pattern modification of the FEP surface was performed by irradiating with an atomic oxygen beam.
メッシュマスクを使用し、原子状酸素を６．０×１０19ａｔｏｍｓ／ｃｍ2照射してパターン改質したＦＥＰ表面における細胞接着パターンの形成過程を図１７に示す。各時刻における観察領域は、それぞれ異なる領域である。細胞播種から２時間後、図１７（ａ）に示すように、改質表面上で細胞がランダムに接着を開始した。播種から９６時間後には、図１７（ｂ）に示すように、細胞密度の密な領域と疎な領域が生じ、破線で示すような輪郭が確認できた。その後、領域内の細胞が増殖し、播種から１２０時間後では、細胞密度の密な領域と疎な領域がさらに広い範囲で顕著に現れ、図１７（ｃ）に示すような細胞接着パターンが確認できた。形成された細胞接着パターンの形状および寸法が、メッシュマスク開口部のそれらとほぼ一致していることから、本方法により、ＦＥＰ表面において、細胞接着パターンが作製可能であることが確認できる。 FIG. 17 shows the process of forming a cell adhesion pattern on the FEP surface that has been subjected to pattern modification by irradiation with atomic oxygen at 6.0 × 10 19 atoms / cm 2 using a mesh mask. The observation areas at each time are different areas. Two hours after cell seeding, cells randomly started to adhere on the modified surface as shown in FIG. 17 (a). After 96 hours from seeding, as shown in FIG. 17 (b), dense and sparse areas of cell density were generated, and an outline as shown by a broken line could be confirmed. Thereafter, cells in the region proliferate, and after 120 hours from seeding, the dense and sparse regions of the cell density remarkably appear in a wider range, confirming the cell adhesion pattern as shown in FIG. did it. Since the shape and dimensions of the formed cell adhesion pattern substantially match those of the mesh mask opening, it can be confirmed that the cell adhesion pattern can be produced on the FEP surface by this method.
本発明は、以上のように構成されており、細胞接着基板材料の基板としてＦＥＰを、比較用基板としてＬＤＰＥを使用し、原子状酸素ビームの照射により表面改質を行い、その改質表面を評価した。改質の結果、ＬＤＰＥ表面では、原子状酸素ビームの照射量に対して、表面粗さには変化はないものの酸素濃度が上昇し、ぬれ性が増大した。一方、ＦＥＰ表面では、原子状酸素ビームの照射による酸素濃度の上昇は見られなかったが、表面に新たな凹凸構造が形成され、表面粗さが増加し、その結果、ぬれ性が低下した。 The present invention is configured as described above, and FEP is used as a cell adhesion substrate material substrate, LDPE is used as a comparative substrate, surface modification is performed by irradiation with an atomic oxygen beam, and the modified surface is formed. evaluated. As a result of the modification, on the LDPE surface, although the surface roughness did not change with respect to the irradiation amount of the atomic oxygen beam, the oxygen concentration increased and the wettability increased. On the other hand, on the FEP surface, the oxygen concentration did not increase due to the irradiation of the atomic oxygen beam, but a new uneven structure was formed on the surface, the surface roughness increased, and as a result, the wettability decreased.
改質表面に骨芽細胞様細胞を播種し、改質表面と細胞との親和性について検討した。その結果、ＬＤＰＥ表面では、改質による有意な接着細胞数の変化が見られなかったが、ＦＥＰ表面では、有意に接着細胞数が増加した。また、ＦＥＰ表面に接着した細胞形状からも、細胞親和性の向上が認められた。 Osteoblast-like cells were seeded on the modified surface, and the affinity between the modified surface and the cells was examined. As a result, there was no significant change in the number of adherent cells due to modification on the LDPE surface, but the number of adherent cells significantly increased on the FEP surface. Moreover, the improvement of cell affinity was recognized also from the cell shape adhere | attached on the FEP surface.
ＦＥＰ表面のパターン改質を行い、改質部と未改質部の細胞親和性の差を利用して細胞接着パターンを作成した。接着パターン形成過程の観察より、初期においては、細胞がランダムに接着を開始し、その後、改質部に接着した細胞が、原子状酸素未照射の基板表面に接着した細胞よりも早く増殖することで、細胞密度に差が生じ、パターンが形成されることがわかった。 The FEP surface was subjected to pattern modification, and a cell adhesion pattern was created by utilizing the difference in cell affinity between the modified part and the unmodified part. From the observation of the adhesion pattern formation process, in the initial stage, cells start to adhere randomly, and then the cells that adhere to the modified part proliferate faster than the cells that adhere to the substrate surface that has not been irradiated with atomic oxygen. Thus, it was found that the cell density was different and a pattern was formed.
また、本発明に係る細胞接着材料は、以上のように、表面構造を物理的に改質することにより、細胞と材料表面との親和性を制御するものであるため、細胞を基板材料から除いた後、再度、原子状ビームにより改質するか、或いは、そのままの状態で基板を細胞接着用材料として再利用することができる可能性も有している。 Moreover, since the cell adhesion material according to the present invention controls the affinity between cells and the material surface by physically modifying the surface structure as described above, the cells are excluded from the substrate material. After that, there is a possibility that the substrate can be modified again with an atomic beam, or the substrate can be reused as a cell adhesion material in the same state.
本発明に係る細胞接着材料は、表面構造を物理的に改質することにより、細胞と材料表面の親和性を制御し、その結果として、細胞接着等の機能を制御できる可能性を示唆するものであり、各種細胞基礎研究用材料および各種医療用材料として産業上の利用価値を有するものである。 The cell adhesion material according to the present invention suggests the possibility of controlling the function of cell adhesion and the like as a result of controlling the affinity between the cell and the material surface by physically modifying the surface structure. It has industrial utility value as various basic cell research materials and various medical materials.
Claims (1)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2003274530A JP4660713B2 (en) | 2003-07-15 | 2003-07-15 | Cell adhesion material |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2003274530A JP4660713B2 (en) | 2003-07-15 | 2003-07-15 | Cell adhesion material |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2005036106A JP2005036106A (en) | 2005-02-10 |
| JP4660713B2 true JP4660713B2 (en) | 2011-03-30 |
Family
ID=34211459
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2003274530A Expired - Fee Related JP4660713B2 (en) | 2003-07-15 | 2003-07-15 | Cell adhesion material |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP4660713B2 (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2016146777A (en) * | 2015-02-12 | 2016-08-18 | ダイキン工業株式会社 | Bag for cell cultivation and cell cultivation method using the same |
| JP6712072B2 (en) * | 2015-02-27 | 2020-06-17 | 学校法人東海大学 | Surface modification and sterilization of cell culture substrate with active oxygen |
| WO2020032109A1 (en) * | 2018-08-08 | 2020-02-13 | 株式会社クレハ | Molded resin object, method for producing molded resin object, and sterilization method |
| JP7409646B2 (en) * | 2020-02-12 | 2024-01-09 | 国立研究開発法人宇宙航空研究開発機構 | Antibacterial molded article and its manufacturing method |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6372100A (en) * | 1986-08-26 | 1988-04-01 | フィジカル・サイエンス・インコ−ポレ−テッド | High flux energy atom source |
| JPS63198981A (en) * | 1987-02-13 | 1988-08-17 | Sumitomo Electric Ind Ltd | Base material for cultivating cell |
| JPH0265867A (en) * | 1988-07-01 | 1990-03-06 | Becton Dickinson & Co | Method for adhering endothelial cell to surface of polymer and article |
| JPH04330277A (en) * | 1991-04-26 | 1992-11-18 | Bio Material Kenkyusho:Kk | Culture substrate |
| JPH04357198A (en) * | 1991-02-20 | 1992-12-10 | Sanyo Electric Co Ltd | Production of superconducting oxide thin film |
| JPH0549689A (en) * | 1991-08-20 | 1993-03-02 | Sony Corp | Cell adhesive material and production thereof |
| JP2004079704A (en) * | 2002-08-14 | 2004-03-11 | New Industry Research Organization | Semiconductor manufacturing device |
-
2003
- 2003-07-15 JP JP2003274530A patent/JP4660713B2/en not_active Expired - Fee Related
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6372100A (en) * | 1986-08-26 | 1988-04-01 | フィジカル・サイエンス・インコ−ポレ−テッド | High flux energy atom source |
| JPS63198981A (en) * | 1987-02-13 | 1988-08-17 | Sumitomo Electric Ind Ltd | Base material for cultivating cell |
| JPH0265867A (en) * | 1988-07-01 | 1990-03-06 | Becton Dickinson & Co | Method for adhering endothelial cell to surface of polymer and article |
| JPH04357198A (en) * | 1991-02-20 | 1992-12-10 | Sanyo Electric Co Ltd | Production of superconducting oxide thin film |
| JPH04330277A (en) * | 1991-04-26 | 1992-11-18 | Bio Material Kenkyusho:Kk | Culture substrate |
| JPH0549689A (en) * | 1991-08-20 | 1993-03-02 | Sony Corp | Cell adhesive material and production thereof |
| JP2004079704A (en) * | 2002-08-14 | 2004-03-11 | New Industry Research Organization | Semiconductor manufacturing device |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2005036106A (en) | 2005-02-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wohlfart et al. | Nanofibrillar patterns by plasma etching: The influence of polymer crystallinity and orientation in surface morphology | |
| Oyane et al. | Simple surface modification of poly (ϵ‐caprolactone) to induce its apatite‐forming ability | |
| Rebollar et al. | Proliferation of aligned mammalian cells on laser-nanostructured polystyrene | |
| Savoji et al. | Electrospun nanofiber scaffolds and plasma polymerization: a promising combination towards complete, stable endothelial lining for vascular grafts | |
| Mozetič | Plasma-stimulated super-hydrophilic surface finish of polymers | |
| Tserepi et al. | Plasma nanotextured polymeric surfaces for controlling cell attachment and proliferation: a short review | |
| Declercq et al. | The role of scaffold architecture and composition on the bone formation by adipose-derived stem cells | |
| CN104039951B (en) | For guiding the device of cell migration and implementing the bootstrap technique of this device | |
| Favia et al. | Novel plasma processes for biomaterials: micro-scale patterning of biomedical polymers | |
| Aflori et al. | Collagen immobilization on polyethylene terephthalate surface after helium plasma treatment | |
| Smirnov et al. | Surface modification of polystyrene thin films by RF plasma treatment | |
| Kenar et al. | Chemical and topographical modification of PHBV surface to promote osteoblast alignment and confinement | |
| Zhu et al. | Effects of laser‐modified polystyrene substrate on CHO cell growth and alignment | |
| Primc et al. | Hydrophobic recovery of plasma-hydrophilized polyethylene terephthalate polymers | |
| Hegemann et al. | Plasma surface engineering for manmade soft materials: A review | |
| CN103865084A (en) | Polyether-ether-ketone material and surface modification method thereof | |
| Fisher | Challenges in the characterization of plasma-processed three-dimensional polymeric scaffolds for biomedical applications | |
| Neděla et al. | Functionalized polyethylene naphthalate for cytocompatibility improvement | |
| Mohsenimehr et al. | Surface modification of PLA scaffold using radio frequency (RF) nitrogen plasma in tissue engineering application | |
| Moyen et al. | Nanostructured conducting polymers for stiffness controlled cell adhesion | |
| Morelli et al. | Utilizing radio frequency plasma treatment to modify polymeric materials for biomedical applications | |
| JP4660713B2 (en) | Cell adhesion material | |
| JP2008306977A (en) | Cell culture substrate | |
| Michelmore et al. | Gradient Technology for High‐Throughput Screening of Interactions between Cells and Nanostructured Materials | |
| Ye et al. | Fabrication of poly HEMA grids by micromolding in capillaries for cell patterning and single‐cell arrays |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20060621 |
|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20080630 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20091215 |
|
| A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20100209 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20100706 |
|
| A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20100811 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20101124 |
|
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20101206 |
|
| R150 | Certificate of patent or registration of utility model |
Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20140114 Year of fee payment: 3 |
|
| S533 | Written request for registration of change of name |
Free format text: JAPANESE INTERMEDIATE CODE: R313533 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
| S531 | Written request for registration of change of domicile |
Free format text: JAPANESE INTERMEDIATE CODE: R313531 |
|
| R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| LAPS | Cancellation because of no payment of annual fees |