JPWO2018021289A1 - 培地用高分子ゲル、培地、細胞の培養方法及びキット - Google Patents
培地用高分子ゲル、培地、細胞の培養方法及びキット Download PDFInfo
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Abstract
Description
項1.可逆的結合によって架橋された架橋構造体と溶媒とを含む、培地用高分子ゲル。
項2.前記可逆的結合が、ホスト基とゲスト基との結合、疎水性相互作用、水素結合、イオン結合、配位結合、π電子相互作用及びこれら以外の分子間相互作用の群から選ばれる少なくとも1種である、項1に記載の培地用高分子ゲル。
項3.前記可逆的結合が、ホスト基とゲスト基との結合である、項1又は2に記載の培地用高分子ゲル。
項4.前記架橋構造体は、
下記一般式(1a)
で表される繰り返し構成単位、
下記一般式(2a)
で表される繰り返し構成単位、及び、
下記一般式(3a)
で表される繰り返し構成単位
を有する重合体である、項1〜3のいずれか1項に記載の培地用高分子ゲル。
項5.項1〜4のいずれか1項に記載の培地用高分子ゲルを含む、培地。
項6.項5に記載の培地で細胞を培養する、細胞の培養方法。
項7.前記培地に前記可逆的結合の形成を阻害する性質を有する競争物質を添加する工程をさらに含む、項6に記載の細胞の培養方法。
項8.前記培地に添加された前記競争物質の濃度を増大又は減少させる、項7に記載の細胞の培養方法。
項9.前記培地に含まれる前記競争物質の濃度を10mol/L以下の範囲に調節する、項7又は8に記載の細胞の培養方法。
項10.項5に記載の培地を含む、キット。
本実施形態に係る培地用高分子ゲルは、可逆的結合によって架橋された架橋構造体と溶媒とを含む。この高分子ゲルは、例えば、後述するように競争物質を存在させること等により、可逆的結合の形成及び切断を制御できるように形成されている。そのため、高分子ゲルの硬さ、すなわち、弾性率等の物性を、動的に制御することが可能な材料である。このように、本実施形態に係る培地用高分子ゲルは、硬さを容易、かつ、可逆的に変化させることができるため、このような培地用高分子ゲルを用いて形成される培地で、細胞を培養すると、高分子ゲルの硬さに応じて、細胞の形および機能を容易に制御させることができる。
本実施形態の培地は、上記した培地用高分子ゲルを含んで構成される。培地用高分子ゲルとは、培地において細胞を培養させるための支持体となり得る基材である。培地は、基材としての当該培地用高分子ゲルを含む限りは、その形態は限定されない。例えば、細胞を培養させるための支持体は、本実施形態の培地用高分子ゲルのみで構成されていてもよいし、他の部材と組み合わせて構成されていてもよい。また、培地は、上記培地用高分子ゲルが培養液に浸されて形成されていてもよい。あるいは、培地は、上記培地用高分子ゲルを減圧脱水又は凍結乾燥等により溶媒を減少又は除去して乾燥状態(例えば、キセロゲル)としたところに、培養液を加えて再度膨潤させて形成させてもよい。
培養の方法は特に限定されず、公知の方法で行うことができる。特に、上記培地は、硬さを制御できる点に特徴があるので、例えば、次のように培地の硬さを変化させつつ、細胞を培養することができる。
図2に示す反応スキームにより、高分子ゲルを調製した。
調製例1で調製した高分子ゲルを、一方に注入口を、その反対側に排出口を備える35mm径のプラスチック製ディッシュ内に固定し、そこにRoswell Park Memorial Institute培地(以下、「RPMI培地」という。)を添加して高分子ゲルを浸漬させ、試験を開始した。実験開始から10分毎に高分子ゲルの弾性率を測定した。なお、弾性率の測定は、原子間力顕微鏡(AFM)により行った。具体的には、カンチレバーを高分子ゲルに押し込み、その際のカンチレバーの変位とひずみに基づいて、弾性率を算出した。
10mMのβ−CDを含むRPMI培地の代わりに2.5mMのβ−CDを含むRPMI培地を用いたこと以外は試験例1と同様にして弾性率を測定した。結果を図3(a)に黒丸で示す。
調製例1で調製した高分子ゲルを、試験例1と同様のプラスチックディッシュ内に固定し、β−CDを含まないRPMI培地中で静置した後、0.5mMのβ−CDを含むRPMI培地を注入口から添加しディッシュ内の溶液を置換し、試験例1と同様にして高分子ゲルの弾性率を測定した。β−CDを1mM、2mM、5mM、10mM、15mM、20mM及び25mM含むRPMI培地を用いて、それぞれ同様に弾性率を測定した。弾性率を縦軸に、β−CD濃度を対数目盛の横軸にプロットした結果を図3(b)に示す。図3(b)に示す結果から、競争物質であるβ−CDの濃度が増えると、架橋構造体中の架橋点が減少することにより、高分子ゲルの弾性率が対数関数的に減少することが明らかである。すなわち、添加するβ−CDの量及び濃度等によって、高分子ゲルの弾性率を所望の値に動的に制御できることが示された。
調製例1で調製した高分子ゲルを、試験例1と同様のプラスチックディッシュ内に固定し、次いで、10%ウシ胎児血清ペニシリン、およびストレプトマイシンを含むRPMI−1640培地(以下、「β−CDを含まないRPMI培地」という。)をディッシュ内に注入し、ここに10000cellsのマウス筋芽細胞(C2C12)を播種し、測定開始まで24時間インキュベーションした。
試験例4の観察と同時に、デジタルデータを2値化処理し、細胞の面積と細胞の真円度を求めた。細胞の面積を図5(a)に、細胞の真円度を図5(b)に示す。なお、細胞の真円度は下記式
(4π×面積)/{(細胞の外周の長さ)^2}
より求め、真円度が1に近いほど円形に近い。
Claims (10)
- 可逆的結合によって架橋された架橋構造体と溶媒とを含む、培地用高分子ゲル。
- 前記可逆的結合が、ホスト基とゲスト基との結合、疎水性相互作用、水素結合、イオン結合、配位結合、π電子相互作用及びこれら以外の分子間相互作用の群から選ばれる少なくとも1種である、請求項1に記載の培地用高分子ゲル。
- 前記可逆的結合が、ホスト基とゲスト基との結合である、請求項1又は2に記載の培地用高分子ゲル。
- 前記架橋構造体は、
下記一般式(1a)
で表される繰り返し構成単位、
下記一般式(2a)
で表される繰り返し構成単位、及び、
下記一般式(3a)
で表される繰り返し構成単位
を有する重合体である、請求項1〜3のいずれか1項に記載の培地用高分子ゲル。 - 請求項1〜4のいずれか1項に記載の培地用高分子ゲルを含む、培地。
- 請求項5に記載の培地で細胞を培養する、細胞の培養方法。
- 前記培地に前記可逆的結合の形成を阻害する性質を有する競争物質を添加する工程をさらに含む、請求項6に記載の細胞の培養方法。
- 前記培地に添加された前記競争物質の濃度を増大又は減少させる、請求項7に記載の細胞の培養方法。
- 前記培地に含まれる前記競争物質の濃度を10mol/L以下の範囲に調節する、請求項7又は8に記載の細胞の培養方法。
- 請求項5に記載の培地を含む、キット。
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WO2018021289A1 (ja) | 2018-02-01 |
US11384334B2 (en) | 2022-07-12 |
EP3492576A4 (en) | 2020-07-15 |
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