JP2019073665A - 接着剤及びその使用 - Google Patents
接着剤及びその使用 Download PDFInfo
- Publication number
- JP2019073665A JP2019073665A JP2017202468A JP2017202468A JP2019073665A JP 2019073665 A JP2019073665 A JP 2019073665A JP 2017202468 A JP2017202468 A JP 2017202468A JP 2017202468 A JP2017202468 A JP 2017202468A JP 2019073665 A JP2019073665 A JP 2019073665A
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- Prior art keywords
- cells
- cell
- embedded
- hydrogel
- adhesive
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Abstract
【解決手段】接着剤は、カチオン性水溶性ポリマーで表面が被覆されたナノ粒子を含む。細胞構造体は、細胞が包埋されたアルギン酸ハイドロゲルと、前記接着剤と、を含む。細胞構造体の製造方法は、アルギン酸ハイドロゲルに細胞を包埋する細胞包埋工程と、複数の前記細胞が包埋された前記アルギン酸ハイドロゲルを前記接着剤を用いて接着する接着工程と、を備える方法である。
【選択図】なし
Description
本発明の第1態様に係る接着剤は、カチオン性水溶性ポリマーで表面が被覆されたナノ粒子を含む。
本発明の一実施形態に係る接着剤は、カチオン性水溶性ポリマーで表面が被覆されたナノ粒子を含む。
図1は、本実施形態の接着剤に含まれるカチオン性水溶性ポリマーで表面が被覆されたナノ粒子(CNP)の一例を示す概略構成図である。
(i)アクリル酸メチル、アクリル酸エチル、アクリル酸イソプロピル、アクリル酸−n−ブチル、アクリル酸−2−エチルヘキシル等のアクリル酸エステル類;
(ii)メタクリル酸メチル、メタクリル酸エチル、メタクリル酸イソプロピル、メタクリル酸−n−ブチル、メタクリル酸イソブチル、メタクリル酸−n−ヘキシル、メタクリル酸シクロヘキシル、メタクリル酸ラウリル、メタクリル酸グリシジル等のメタクリル酸エステル類;
(iii)スチレン、α−メチルスチレン等の芳香族オレフィン;
(iv)酢酸ビニル等のビニルエステル;
(v)アクリロニトリル、メタアクリロニトリロ等のビニルニトリル。
本実施形態の接着剤に含まれるCNPは、上記カチオン性モノマーと上記疎水性モノマーとを水溶媒中でラジカル重合開始剤の存在下において乳化重合させることで得られる。
(i)2,2’−アゾビスイソブチロニトリル、2,2’−アゾビス(2,4−ジメチルバレロニトリル等の油溶性アゾ化合物;
(ii)2,2’−アゾビス(2−アミジノプロパン)若しくはその塩酸塩、4,4’−アゾビス(4−シアノバレリン酸)若しくはそのアルカリ金属塩、2,2’−アゾビス[2−(2−イミダゾリン−2−イル)プロパン]若しくはその塩酸塩、2,2’−アゾビス[2−メチル−N−(2−ヒドロキシエトル)プロピオンアミド]等の水溶性アゾ化合物;
(iii)ベンゾイルオキシパーオキサイド、ジターシャリイブチルパーオキサイド等の有機過酸化物;
(iv)過硫酸カリウム、過硫酸ナトリウム、過硫酸アンモニウム等の無機過酸化物;
(v):上記(iv)と還元性物質(亜硫酸ナトリウム、ジメチルアミノエタノール、ジメチルアミノ安息香酸等)とを組み合わせたレドックス開始剤。
−イミダゾリン−2−イル)プロパン]若しくはその塩酸塩、又は、2,2’−アゾビス[2−メチル−N−(2−ヒドロキシエトル)プロピオンアミド]であることが好ましい。
(i)カチオン性モノマー、疎水性モノマー及びラジカル重合開始剤を一括して反応槽に仕込んで重合を行う方法;
(ii)カチオン性モノマー、疎水性モノマー及びラジカル重合開始剤をそれぞれ個別に滴下しつつ重合する方法;
(iii)疎水性モノマー及びラジカル重合開始剤の混合物を(カチオン性モノマー)を含む水中に滴下しつつ重合する方法。
本実施形態の接着剤は、粉末状であってもよく、液体状であってもよい。また、本実施形態の接着剤は、CNP以外に、CNPのカチオン性を損なわない程度に、その他の成分を含んでいてもよい。その他の成分としては、例えば、安定剤、増粘剤、防腐剤等が挙げられる。
本実施形態の接着剤は、上記CNPと、必要に応じて、その他成分とを混合することで得られる。
本実施形態の接着剤は、アニオン性水溶性物質の接着に好適に用いられる。
本発明の一実施形態に係る細胞構造体は、細胞が包埋されたアルギン酸ハイドロゲルと、上記接着剤と、を含む。
本実施形態の細胞構造体に含まれる細胞としては、特別な限定はなく、例えば、生殖細胞(精子、卵子等)、生体を構成する体細胞、幹細胞、前駆細胞、生体から分離されたがん細胞、生体から分離され不死化能を獲得して体外で安定して維持される細胞(細胞株)、生体から分離され人為的に遺伝子改変された細胞、生体から分離され人為的に核が交換された細胞等が挙げられ、これらに限定されない。また、これら細胞の細胞塊(スフェロイド)を用いてもよい。また、生体の正常組織又はがん組織から分離された組織片を、そのまま用いてもよい。
本実施形態の細胞構造体は、細胞が包埋されたアルギン酸ハイドロゲル及び上記接着剤の他に、さらに、その他の成分を含んでいてもよい。
本発明の一実施形態に係る細胞構造体の製造方法は、細胞包埋工程と、接着工程と、を備える方法である。
細胞包埋工程は、アルギン酸ハイドロゲルに細胞を包埋する工程である。
接着工程は、複数の細胞が包埋されたアルギン酸ハイドロゲルを上記接着剤を用いて接着する工程である。
洗浄工程は、上記細胞包埋工程において得られた細胞が包埋されたアルギン酸ハイドロゲルを洗浄するために行ってもよい。又は、上記接着工程において得られた細胞構造体を洗浄するために行ってもよい。
除去工程は、細胞構造体に含まれるアルギン酸ハイドロゲルを除去するための工程である。これにより、細胞同士が接着剤を介して直接接着された細胞構造体を得ることができる。
加熱、冷却及び撹拌可能な反応器に、窒素雰囲気下で、水160g、ポリエチレンイミン(MERCK社製、分子量:60万〜100万、50v/w%水溶液)300μL、及び、スチレン8.5gを仕込み撹拌した。次いで、重合開始剤として、ペルオキソ二硫酸アンモニウム(関東化学社製)を25μg添加し、窒素雰囲気下、70℃の条件で2時間撹拌しながら重合反応を行った。重合に伴い、重合系は次第に白濁し乳化状態となった。重合終了後、均一なエマルションとして、ポリエチレンイミンが表面を被覆し、中心部がポリスチレンからなるカチオン性ナノ粒子(以下、「CNP」と略記する場合がある)が得られた。得られたCNPの走査型電子顕微鏡(SEM)像を図3Aに示す。なお、スケールバーは50nmである。図3Aから、CNPは球状であることが確かめられた。
1.板状アルギン酸ハイドロゲルの準備
3.0質量%のアルギン酸ナトリウム水溶液と、0.2質量%の炭酸カルシウム水溶液と、該炭酸カルシウムに対して2倍mol量のグルコノラクトンとを混合して、板状のアルギン酸ハイドロゲル(縦20mm×横5mm×厚さ1mm)を2枚作製した。そのうち1枚については、切断し、製造例1で得られたCNPを塗布して接着させた。
図4Aに示すように、「1.」で得られた1枚のアルギン酸ハイドロゲル(single plate)及びCNPで接着したアルギン酸ハイドロゲル(connected plate)の上下をそれぞれアンカー及びクランプで挟み込み、引っ張り試験機(Instron TE200N)を用いて、引っ張り試験を行った。それぞれの引っ張り挙動を図4Bに示す。図4Bにおいて、左のグラフは、アルギン酸ハイドロゲル(single plate)の引っ張り挙動を示し、右のグラフはCNPで接着したアルギン酸ハイドロゲル(connected plate)の引っ張り挙動を示す。
1.HeLa細胞の準備
予め、6ウェルプレートに4.0×104cells/ウェルとなるようにHeLa細胞を播種した。
次いで、10mg/mLのCNPを含む培地、又は、対照として10mg/mLのポリエチレンイミン(PEI)を含む培地に交換し、37℃で1分間培養した。1分後、Calcein−AM(生細胞を染色)及びEthmdium homodimer(死細胞を染色)を用いて細胞を染色し、細胞生存率を算出した。細胞の染色像を図5A(上:CNP含有培地、下:PEI含有培地)に示す。なお、スケールバーは200μmである。また、各細胞生存率を図5Bに示す。なお、図5Bにおいて、「None」とはCNP及びPEIを添加せずただの培地中にて培養した細胞を示す。
1.蛍光物質を包埋した繊維状アルギン酸ハイドロゲルの作製
参考文献1(H. Onoe et al., “Metre-long cell-laden microfibres exhibit tissue morphologies and functions”, Nature Materials, Vol.12, p584-590, 2013.)に記載の方法を用いて、蛍光物質を包埋した繊維状アルギン酸ハイドロゲルを調製した。なお、蛍光物質としては、以下のものをそれぞれ包埋した。
赤色:FluoSpheres、carboxylate−modified 0.2μm、red(励起波長:580nm、蛍光波長:605nm)
緑色:FluoSpheres、carboxylate−modified 0.2μm、yellow−green(励起波長:505nm、蛍光波長:515nm)
青色:FluoSpheres、carboxylate−modified 0.2μm、blue Fluorescent(励起波長:365nm、蛍光波長:415nm)
次いで、「1.」で得られた蛍光物質を包埋した各繊維状アルギン酸ハイドロゲルにCNPを塗布し、接着してバンドル化した。バンドル化した蛍光物質を包埋した繊維状アルギン酸ハイドロゲルの蛍光顕微鏡(オリンパス社製、IX71)で撮影した画像を図6Aに示す。図6Aにおいて、左上は各色の蛍光物質を包埋した繊維状アルギン酸ハイドロゲルをバンドル化したものである。左下は、青色蛍光物質を包埋した繊維状アルギン酸ハイドロゲルをバンドル化したものである。右上は、緑色蛍光物質を包埋した繊維状アルギン酸ハイドロゲルをバンドル化したものである。右下は、赤色蛍光物質を包埋した繊維状アルギン酸ハイドロゲルをバンドル化したものである。なお、スケールバーは200μmである。
さらに、バンドル化した蛍光物質を包埋した繊維状アルギン酸ハイドロゲルをアルギン酸ハイドロゲルからなる支持材の中に包埋した。次いで、支持材とともにバンドルをミクロトーム用ブレードを2つ重ねたものを用いて、スライスした。スライスの厚みは200μm程度であった。得られたスライスについて、蛍光顕微鏡(オリンパス社製、IX71)で撮影した画像を図6Bに示す。図6Bにおいて、「シェルなし」とは、蛍光物質を包埋した繊維状アルギン酸ハイドロゲルを示す。また、「シェルあり」とは、蛍光物質を包埋した繊維状アルギン酸ハイドロゲルの周囲をさらに蛍光物質を包埋していないアルギン酸ハイドロゲルで覆い、層分離構造を有するものを示す。なお、右側の画像は左側の画像をそれぞれ拡大したものである。また、左側の画像のスケールバーは200μmである。右側の画像のスケールバーはそれぞれ100μmである。
また、図6Bから、得られたバンドル化した蛍光物質を包埋した繊維状アルギン酸ハイドロゲルを容易にスライス化できることが確かめられた。
1.細胞を包埋した繊維状アルギン酸ハイドロゲルの作製
上記参考文献1に記載の方法を用いて、細胞を包埋した繊維状アルギン酸ハイドロゲルを調製した。なお、細胞としては、6.0×107cellscells/mLのヒト皮膚線維芽細胞の懸濁液を用いた。また、細胞は予めCalcein−AM(生細胞を染色)、Ethmdium homodimer(死細胞を染色)及びHoechst(核を染色)を用いて染色しておいた。
次いで、「1.」で得られた細胞を包埋した繊維状アルギン酸ハイドロゲルにCNPを塗布し、接着してバンドル化した。次いで、バンドル化した細胞を包埋した繊維状アルギン酸ハイドロゲルをアルギン酸ハイドロゲルからなる支持材の中に包埋した。次いで、支持材とともにバンドルをミクロトーム用ブレードを2つ重ねたものを用いて、スライスした。スライスの厚みは200μm程度であった。得られたスライスについて、蛍光顕微鏡(オリンパス社製、IX71)で撮影した画像を図7に示す。なお、スケールバーはそれぞれ100μmである。
1.3Dプリンターを用いた細胞包埋繊維状アルギン酸ハイドロゲルの三次元構造体の作製
図8Aに記載の3Dプリンター(本発明者ら作製)を用いて、細胞包埋繊維状アルギン酸ハイドロゲルの三次元構造体を作製した。具体的な製造工程は図8Bに示すとおりである。まず、3Dプリンターを用いて、6.0×107cells/mLのヒト皮膚線維芽細胞及びECM含有溶液と、2質量%アルギン酸ナトリウム溶液と、10mM塩化カルシウム溶液とを、図8Bに記載の三次元構造となるように、プリントした。次いで、培地で2〜3回洗浄した。次いで、培地を除去して、37℃、5%CO2環境下で30分間インキュベートした。
次いで、製造例1で得られたCNPを含む溶液に細胞包埋繊維状アルギン酸ハイドロゲルの三次元構造体を30秒間浸漬した。
次いで、クエン酸溶液に細胞包埋繊維状アルギン酸ハイドロゲルの三次元構造体を3分間浸漬して、アルギン酸ハイドロゲルを除去した。これにより、アルギン酸ハイドロゲルが除去され、細胞及びECMを含有する三次元的に形成された細胞構造体を得ることができた。
また、「2.」で得られたCNPで接着された細胞包埋繊維状アルギン酸ハイドロゲルの三次元構造体を、培地を含む10cmディッシュに入れて、振盪撹拌機(NISSIN社製、NA−301)を用いて、37℃、5%CO2環境下で5日間インキュベートした。また、対照として、CNPで接着された細胞包埋繊維状アルギン酸ハイドロゲルの三次元構造体を、培地を含む10cmディッシュに入れて、37℃、5%CO2環境下で5日間静置培養した。それぞれの細胞構造体の経時変化を図8Cに示す。
Claims (6)
- カチオン性水溶性ポリマーで表面が被覆されたナノ粒子を含む接着剤。
- 前記カチオン性水溶性ポリマーがポリエチレンイミン又はその塩である請求項1に記載の接着剤。
- アルギン酸ハイドロゲル又は細胞組織の接着に用いられる請求項1又は2に記載の接着剤。
- 前記アルギン酸ハイドロゲルの内部に細胞外マトリックス及び細胞からなる群より選択される少なくとも1種を含む請求項3に記載の接着剤。
- 細胞が包埋されたアルギン酸ハイドロゲルと、
請求項3又は4に記載の接着剤と、
を含む細胞構造体。 - アルギン酸ハイドロゲルに細胞を包埋する細胞包埋工程と、
複数の前記細胞が包埋された前記アルギン酸ハイドロゲルを請求項3又は4に記載の接着剤を用いて接着する接着工程と、
を備える細胞構造体の製造方法。
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