JPWO2018131673A1 - 新規組織再生材料およびその製造方法 - Google Patents
新規組織再生材料およびその製造方法 Download PDFInfo
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- A—HUMAN NECESSITIES
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
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- A—HUMAN NECESSITIES
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
- A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
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Abstract
Description
a)培養環境の制約から幹細胞の分化能などのポテンシャルが最大限活かされていない。
b)組織再生材料の力学的強度が低く、また周囲組織と癒合能が低い。
c)調製可能な組織再生材料が小体積のため広領域の治療に用いることができない。
(1)幹細胞を含む中性の培養培地と、コラーゲン溶液または分散体とを混合する;
(2)(1)の混合物を遠心分離して、幹細胞とコラーゲンを集積させる;
(3)幹細胞とコラーゲンの集積物を培養する;
を含む、前記方法。
一態様において本発明は以下の工程:
(1)幹細胞を含む中性の培養培地と、コラーゲン溶液または分散体とを混合する;
(2)(1)の混合物を遠心分離して、幹細胞とコラーゲンを集積させる;
(3)幹細胞とコラーゲンの集積物を培養する;
を含む、組織再生材料の製造方法に関する。
一態様において本発明は、以下の工程:
(1)幹細胞を含む中性の培養培地と、コラーゲン溶液または分散体とを混合する;
(2)(1)の混合物を遠心分離して、幹細胞とコラーゲンを集積させる;
(3)幹細胞とコラーゲンの集積物を培養する;
を含み、ここで上記(1)または(3)の工程においてアスコルビン酸もしくはアスコルビン酸誘導体またはそれらの塩を添加することを含む方法、により製造される組織再生材料に関する。
(1)幹細胞/コラーゲン複合体の作製
ヒト滑膜より採取した間葉系幹細胞(MSCs)を、培養培地(DMEM、10%ウシ胎児血清(FBS)、1%P/S(ペニシリン/ストレイプトマイシン)中で、4.0×105細胞/cm2または2.0×105細胞/cm2となるまで培養した。培養した細胞をチューブに集め、培養培地(DMEM、10%FBS、1%P/S、0.2mMアスコルビン酸2−リン酸)中、5.6×105細胞/mLまたは2.8×105細胞/mLの細胞懸濁液となるよう調製した。
上記(1)において、2.8×105細胞/mLの細胞懸濁液を用いて作製した幹細胞/コラーゲン複合体を用い、特許文献1の方法に従って作製したTECとの比較を走査型電子顕微鏡(SEM)画像解析により行った。結果を図2に示す。
実施例1(1)では、コラーゲン線維/MSCs溶液を6ウェルプレートにおいて遠心分離を行った。実施例2においては、この遠心分離工程のない実験により得られる組織と、実施例1(1)の方法による幹細胞/コラーゲン複合体を比較した。また、下記実施例4において示すコラーゲンゲル内培養で生成される組織と、実施例1(1)の方法による幹細胞/コラーゲン複合体を比較した。
幹細胞/コラーゲン複合体生成におけるアスコルビン酸2−リン酸添加の影響を検討すべく、以下の方法で幹細胞/コラーゲン複合体を調製した。
ヒト滑膜より採取した間葉系幹細胞(MSCs)を、培養培地(DMEM、10%FBS、1%P/S(ペニシリン/ストレイプトマイシン)中で、2.0×105細胞/cm2となるまで培養した。培養した細胞をチューブに集め、培養培地(DMEM、10%FBS、1%P/S、0.2mMアスコルビン酸2−リン酸)中、2.8×105細胞/mLの細胞懸濁液となるよう調製した。
上記の培養1日目にアスコルビン酸2−リン酸(0.2mM)を添加した群、培養4日目にアスコルビン酸2−リン酸(0.2mM)を添加した群、およびアスコルビン酸2−リン酸を添加しない群はともに、シート状の幹細胞/コラーゲン複合体を形成した。
上記(1)において、2.8×105細胞/mLの細胞懸濁液を用いて作製した幹細胞/コラーゲン複合体を用い、以下に示すコラーゲンゲル内培養によって作製した組織との形態学的な比較を、肉眼による形態観察およびSEM画像解析により行った。
コラーゲン線維とともに細胞培養を行う一般的な方法として、コラーゲンゲル内培養が知られている。非特許文献4の記載に基づき、以下の方法により、組織を調製した。
結果を図4に示す(「コラーゲンゲル内培養」と「細胞/コラーゲン複合体(アスコルビン酸2リン酸有り」の画像)。実施例2において言及したとおり、コラーゲンゲル内培養では大きいものの、ピンセットで把持すると破壊しそうな、多量の水を含む非常に弱い組織が生じた。一方、実施例1(1)の方法により作製した幹細胞/コラーゲン複合体は、強固なシート状の組織であった。
Claims (7)
- 組織再生材料の製造方法であって、以下の工程:
(1)幹細胞を含む中性の培養培地と、コラーゲン溶液または分散体とを混合する;
(2)(1)の混合物を遠心分離して、幹細胞とコラーゲンを集積させる;
(3)幹細胞とコラーゲンの集積物を培養する;
を含む、前記方法。 - 幹細胞が、間葉系幹細胞、ES細胞、または人工多能性幹細胞(iPS細胞)である、請求項1に記載の方法。
- 工程(1)の幹細胞が、1.0×105細胞/cm2〜6.0×105細胞/cm2の細胞密度まで培養した幹細胞である、請求項1または2に記載の方法。
- コラーゲンが、I型、II型またはIII型コラーゲンである、請求項1〜3のいずれか1項に記載の方法。
- 工程(1)のコラーゲン溶液または分散体が、再線維化コラーゲンの線維分散体である、請求項1〜4のいずれか1項に記載の方法。
- 工程(1)または工程(3)においてアスコルビン酸もしくはアスコルビン酸誘導体またはそれらの塩を添加することを含む、請求項1〜5のいずれか1項に記載の方法。
- 請求項1〜6のいずれか1項に記載の方法により製造される、組織再生材料。
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