JP7461382B2 - 抗炎症剤、及び抗炎症作用を有する細胞外小胞の製造方法 - Google Patents
抗炎症剤、及び抗炎症作用を有する細胞外小胞の製造方法 Download PDFInfo
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Description
(1)間葉系幹細胞に由来する細胞外小胞からホスファチジルセリンに対して親和性を有する物質を利用する方法により得られる細胞外小胞を有効成分とする、抗炎症剤;
(2)前記間葉系幹細胞が、iPS細胞に由来するもの、或いは臍帯、臍帯血、骨髄、脂肪、筋肉、神経、皮膚、羊膜及び胎盤からなる群より選択される1種以上の組織に由来するものである上記(1)に記載の抗炎症剤;
(3)ホスファチジルセリンに対する親和性を有する物質が、Timタンパク質である上記(1)又は(2)に記載の抗炎症剤;
(4)Timタンパク質が、Tim4タンパク質、Tim3タンパク質及びTim1タンパク質から選択されるものである上記(3)に記載の抗炎症剤;
である。
(5)間葉系幹細胞由来の細胞外小胞からホスファチジルセリンに対して親和性を有する物質を用いる方法により細胞外小胞を得ることを含む、抗炎症作用を有する細胞外小胞の製造方法;
(6)ホスファチジルセリンに対する親和性を有する物質が、Timタンパク質である上記(5)に記載の細胞外小胞の製造方法;
(7)Timタンパク質が、Tim4タンパク質、Tim3タンパク質及びTim1タンパク質から選択されるものである上記(6)に記載の細胞外小胞の製造方法;
である。
(1)カルシウムイオン存在下、EVを含む細胞培養上清液とPS親和性物質とを接触させて、当該細胞培養上清液中のPS陽性細胞外小胞とPS親和性物質との複合体(本発明に係る複合体)を形成させること(以下、「複合体形成工程」と略記する場合がある)、
(2)前記EVを含む細胞培養上清液から、複合体形成工程で得られた本発明に係る複合体を分離すること(以下、「複合体分離工程」と略記する場合がある)、
(3)本発明に係る複合体からPS陽性細胞外小胞を分離し、PS陽性細胞外小胞を取得すること(以下、「取得工程」と略記する場合がある)。
(1)細胞培養
骨髄由来間葉系幹細胞であるPoieticsTM ヒト間葉系幹細胞(LONZA社)を、15%FBS(Selborne Biological Sevice Pty.社)含有MEMα(L-グルタミン、フェノールレッド含有、富士フイルム和光純薬(株))を増殖培地として用いて培養した。その後、培養した骨髄由来間葉系幹細胞を細胞数3×105で100mm細胞培養用ディッシュ(Corning International社)に播種し、5%CO2、37℃の条件に設定した細胞培養用インキュベータ内で72時間培養し、細胞数3×106まで増殖させた。
(2)細胞外小胞の産生
(1)で増殖させた骨髄由来間葉系幹細胞を、細胞外小胞産生培地である10%GIBCO:Fetal Bovine Serum, exosome-depleted, One ShotTM format(Thermo Fisher Scientific社)含有D-MEM(富士フイルム和光純薬(株))20mLに置換し、5%CO2、37℃の条件に設定した細胞培養用インキュベータ内で120時間培養を行った。その後、得られた培養上清を50mLの遠沈管に回収して2,000×gで20分間遠心し、上清を回収した。
(3)PSアフィニティー法による細胞外小胞の取得
(2)で回収した培養上清1mLから、MagCaptureTM Exosome Isolation Kit PS(富士フイルム和光純薬(株))を用いて、当該キットに添付の説明書に記載の手順に従って細胞外小胞を単離し、EV-SaveTM 細胞外小胞ブロッキング試薬(富士フイルム和光純薬(株))を添加した1mM EDTA含有PBS(Phosphate-buffered saline)中に細胞外小胞を得た。その後、ビバスピン500(ザルトリウス社、分画分子量:100,000(100K)、膜材質:PES)を用いて、EV-SaveTM 細胞外小胞ブロッキング試薬を添加したPBSへバッファー交換を行った。以下、得られた溶液を「細胞外小胞溶液(PSアフィニティー法、骨髄由来MSC)」と記載する場合がある。
実施例1(1)-(2)と同様の方法に従って調製した培養上清を、超遠心分離機(Beckman:OptimaTM L-100XP)を用いて110,000×gで70分間遠心分離した。得られたペレットに1mLのPBSを添加し、再度110,000×gで70分間遠心分離した。最終的に得られたペレットをEV-SaveTM 細胞外小胞ブロッキング試薬(富士フイルム和光純薬(株))を添加したPBSにより溶解した。以下、得られた溶液を、「細胞外小胞溶液(超遠心法、骨髄由来MSC)」と記載する場合がある。
実施例1で得られた「細胞外小胞溶液(PSアフィニティー法、骨髄由来MSC)」の単位体積当たりの粒子数を、NanoSight(Malvern Panalytical社)を用いて、ナノ粒子トラッキング解析法(Nano Tracking Analysis法)により、NanoSightの説明書に記載の手順に従って測定し、平均粒子径と単位体積当たりの平均粒子数[particles/mL]を算出した。得られた粒径分布のグラフを実験例2の結果と併せて図1に示す。図1のグラフにおいて、縦軸は粒子数、横軸は粒径をそれぞれ示す。PSアフィニティー法により取得した細胞外小胞の平均粒径は、138.1±0.2nm、単位体積当たりの平均粒子数は、1.76×1010[particles/mL]であった。
「細胞外小胞溶液(PSアフィニティー法、骨髄由来MSC)」の代わりに、比較例1で得られた「細胞外小胞溶液(超遠心法、骨髄由来MSC)」を用いた以外は、実験例1と同様の方法により、平均の粒子径と単位体積当たりの平均粒子数[Particles/mL]を算出した。得られた粒径分布のグラフを実験例1の結果と併せて図1に示す。超遠心法により取得した細胞外小胞の平均粒径は、138.1±2.9nm、単位体積当たりの平均粒子数は、1.68×1010[particles/mL]であった。
実施例1で得られた「細胞外小胞溶液(PSアフィニティー法、骨髄由来MSC)」を用いて、細胞外小胞マーカータンパク質であるCD9、CD63、およびCD81についてウエスタンブロット法により解析した。
実験例1で算出した「細胞外小胞溶液(PSアフィニティー法、骨髄由来MSC)」中の単位体積当たりの平均粒子数に基づいて、3.0×108 particles分の「細胞外小胞溶液(PSアフィニティー法、骨髄由来MSC)」に1/4量のSDS-PAGE Sample Buffer 4倍濃縮液(還元剤不含)(富士フイルム和光純薬(株))を混合し、全量を用いて10-20%アクリルアミドゲル(富士フイルム和光純薬(株))により電気泳動した。その後、転写バッファー(富士フイルム和光純薬(株))を用いて、PVDFメンブレン(BioRad社)に転写した。転写を行ったメンブレンは、1%スキムミルク含有PBS-T溶液(0.1(w/v)% TweenTM20含有PBS溶液)に1時間浸してブロッキング処理を行い、抗CD9抗体(富士フイルム和光純薬(株))、抗CD63抗体(富士フイルム和光純薬(株))、および抗CD81抗体(富士フイルム和光純薬(株))をそれぞれPBS-T溶液で1.1μg/mLに調整した一次抗体溶液と反応させた。その後、抗CD9抗体、抗CD81抗体を反応させたメンブレンは、HRP標識抗ラットIgG抗体(Jackson Immuno Research社)をPBS-T溶液で10000倍希釈した二次抗体溶液と反応させ、抗CD63抗体を反応させたメンブレンは、HRP標識抗マウスIgG抗体(DAKO社)PBS-T溶液で10000倍希釈した二次抗体溶液とそれぞれ反応させた。その後、検出試薬としてイムノスターTM ゼータ(富士フイルム和光純薬(株))を使用し、Amersham Imager 600(GEヘルスケア社)を用いてシグナルを検出した。ウエスタンブロットの結果を、実験例4の結果と併せて図2に示す。図中、横軸のPSは「細胞外小胞溶液(PSアフィニティー法、骨髄由来MSC)」を用いた場合の結果(実験例3)、UCは「細胞外小胞溶液(超遠心法、骨髄由来MSC)」を用いた場合の結果(実験例4)をそれぞれ示す。また、縦軸の矢印は、検出した細胞外小胞マーカータンパク質のバンド位置を示す。
「細胞外小胞溶液(PSアフィニティー法、骨髄由来MSC)」の代わりに、比較例1で得られた「細胞外小胞溶液(超遠心法、骨髄由来MSC)」を用いた以外は、実験例3と同様の方法により、細胞外小胞マーカータンパク質であるCD9、CD63、およびCD81についてウエスタンブロット法により解析した。ウエスタンブロットの結果を、実験例3の結果と併せて図2に示す。
実施例1で調製した「細胞外小胞溶液(PSアフィニティー法、骨髄由来MSC)」中の細胞外小胞の抗炎症作用を評価した。
10%FBS(Biosera社)含有RPMI(富士フイルム和光純薬(株))にヒト末梢血単核球であるPBMC(peripheral blood mononuclear cell富士フイルム和光純薬(株))を懸濁し、48well plate(ThermoFisher Scientific社)に1ウェルあたり細胞数5×105、培地量250μLでそれぞれ播種した。細胞播種から2時間後、PBS(Phosphate-buffered saline、富士フイルム和光純薬(株))250μLにより細胞を洗浄して非接着細胞を除去した。
「細胞外小胞溶液(PSアフィニティー法、骨髄由来MSC)」の代わりに、比較例1で得られた「細胞外小胞溶液(超遠心法、骨髄由来MSC)」を用いた以外は、実施例2-3と同様の方法により、炎症性サイトカインであるTNFα(比較例2)、IL-6(比較例3)、および内部標準であるGAPDHのmRNA発現量を、それぞれ定量PCRにより測定し、細胞外小胞の抗炎症作用を評価した。定量PCRの結果を実施例2-4の結果と併せて、それぞれ図4-5に示す。
「骨髄由来間葉系幹細胞であるPoieticsTM ヒト間葉系幹細胞(LONZA社)」の代わりに、「臍帯由来間葉系幹細胞であるHuman Mesenchymal Stem Cells from Umbilical Cord Matrix(PromoCell社)」(実施例5)又は「脂肪由来間葉系幹細胞であるHuman Adipose-Derived Stem Cells(LONZA社)」(実施例6)を用いた以外は、実施例1と同様の方法により細胞外小胞の取得を行い、「細胞外小胞溶液(PSアフィニティー法、臍帯由来MSC)」(実施例5)および「細胞外小胞溶液(PSアフィニティー法、脂肪由来MSC)」(実施例6)をそれぞれ得た。
「骨髄由来間葉系幹細胞であるPoieticsTM ヒト間葉系幹細胞(LONZA社)」の代わりに、「臍帯由来間葉系幹細胞であるHuman Mesenchymal Stem Cells from Umbilical Cord Matrix(PromoCell社)」(比較例4)又は「脂肪由来間葉系幹細胞であるHuman Adipose-Derived Stem Cells(LONZA社)」(比較例5)をそれぞれ用いた以外は、実施例1(1)-(2)と同様の方法に従って調製した培養上清を、超遠心分離機(Beckman:OptimaTM L-100XP)を用いて110,000×gで70分間遠心分離した。得られたペレットに1mLのPBSを添加し、再度110,000×gで70分間遠心分離した。最終的に得られたペレットをEV-SaveTM 細胞外小胞ブロッキング試薬(富士フイルム和光純薬(株))を添加したPBSにより溶解し、「細胞外小胞溶液(超遠心法、臍帯由来MSC)」(比較例4)および「細胞外小胞溶液(超遠心法、脂肪由来MSC)」(比較例5)をそれぞれ得た。
実施例5及び6で調製した「細胞外小胞溶液(PSアフィニティー法)」中の細胞外小胞の抗炎症作用をそれぞれ評価した。10%FBS(Biosera社)含有RPMI(富士フイルム和光純薬(株))にヒト末梢血単核球であるPBMC(peripheral blood mononuclear cell、富士フイルム和光純薬(株))を懸濁し、96well plate(ThermoFisher Scientific社)に1ウェルあたり細胞数4×105、培地量100μLでそれぞれ播種した。細胞播種から2時間後、PBS(Phosphate-buffered saline、富士フイルム和光純薬(株))200μLにより細胞を洗浄して非接着細胞を除去した。
「細胞外小胞溶液(PSアフィニティー法)」の代わりに、比較例4で得られた「細胞外小胞溶液(超遠心法、臍帯由来MSC)」(比較例6-8)又は比較例5で得られた「細胞外小胞溶液(超遠心法、脂肪由来MSC)」(比較例9)を用いた以外は、実施例7-12と同様の方法により、炎症性サイトカインであるTNFα(比較例6)、IL-6(比較例7)、TGFβ1(比較例8及び9)並びに内部標準であるGAPDHのmRNA発現量を、それぞれ定量PCRにより測定し、細胞外小胞の抗炎症作用を評価した。 定量PCRの結果を実施例7-12の結果と併せて、それぞれ図7-9に示す。
Claims (3)
- 間葉系幹細胞由来の細胞外小胞からTimタンパク質を用いる方法により細胞外小胞を得ることを含む、抗炎症作用を有する細胞外小胞の製造方法。
- Timタンパク質が、Tim4タンパク質、Tim3タンパク質及びTim1タンパク質から選択されるものである、請求項1に記載の細胞外小胞の製造方法。
- 請求項1又は2に記載の細胞外小胞の製造方法により得られた細胞外小胞を有効成分として含有させることを含む、抗炎症剤の製造方法。
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