JP6637656B2 - 角膜内皮細胞を含有する移植用組成物 - Google Patents
角膜内皮細胞を含有する移植用組成物 Download PDFInfo
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- JP6637656B2 JP6637656B2 JP2015005492A JP2015005492A JP6637656B2 JP 6637656 B2 JP6637656 B2 JP 6637656B2 JP 2015005492 A JP2015005492 A JP 2015005492A JP 2015005492 A JP2015005492 A JP 2015005492A JP 6637656 B2 JP6637656 B2 JP 6637656B2
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Description
(1)エピガロカテキンガレートを含有する溶液中にヒト角膜内皮細胞を含んでなる,角膜内皮機能不全患者にヒト角膜内皮細胞を移植するために調製された,組成物。
(2)該エピガロカテキンガレートの濃度が15〜35μg/mLである,上記(1)の組成物。
(3)該エピガロカテキンガレートの濃度が25μg/mLである,上記(1)の組成物。
(4)該ヒト角膜内皮細胞の濃度が,1×106〜4×106個/mLである,上記(1)〜(3)の何れかの組成物。
(5)該ヒト角膜内皮細胞の濃度が, 2.5x106個/mLである,上記(1)〜(3)の何れかの組成物。
(6)該角膜内皮機能不全患者が,水疱性角膜症,角膜浮腫,角膜白斑及び円錐角膜からなる群から選択される何れかの疾患に罹患しているものである,上記(1)〜(5)の何れかの組成物。
(7)一つの眼球あたり,1回に1×105〜8×105個の該ヒト角膜内皮細胞が投与されるものである,上記(1)〜(6)の何れかの組成物。
(8)該調製後に,凍結されることなく該移植に用いられるものである,上記(1)〜(7)の何れかの組成物。
(9)該ヒト角膜内皮細胞が,ヒト角膜組織から得たヒト角膜内皮細胞を,間葉系幹細胞の馴化培地からなる又は該馴化培地を含有する,角膜内皮細胞培養用培地を用いて培養して増殖させたものである,上記(1)〜(8)の何れかの組成物。
(10)該角膜内皮細胞培養用培地が,該馴化培地を0.5〜50%(v/v)の比率で含有するものである,上記(9)の組成物。
(11)該角膜内皮細胞培養用培地が,該馴化培地を3〜10%(v/v)の比率で含有するものである,上記(9)の組成物。
(12)該馴化培地が,該間葉系幹細胞を,コンドロイチン硫酸又はその塩を0.04〜0.12%(w/v)濃度で含有する培地で培養することにより得られたものである,上記(9)〜(11)の何れかの組成物。
(13)該コンドロイチン硫酸又はその塩の濃度が0.08%(w/v)である,上記(12)の組成物。
ヒト骨髄由来のヒト間葉系幹細胞を,5,000〜10,000 個/cm2の密度となるように150mm細胞培養用プレート(コーニング社製)に播種し,10% FCS/DMEM培地を用いてサブコンフルエントになるまで培養した。細胞を,PBS(-)で2回洗浄した後,トリプシン処理して細胞を細胞培養用プレートから剥離し,次いで10% FCS/DMEM培地を添加して懸濁した後,遠心(1200rpm,5分)して細胞を回収した。細胞を10% FCS/DMEM培地で懸濁して,3,000〜5,000個/cm2の密度となるように150mm細胞培養用プレート(コーニング社製)に播種し,細胞密度がプレート底面に対し50〜70%になるまで培養した。培地を捨て,8% FCS,200μg/mL CaCl2・2H2O,0.08%(w/v) コンドロイチン硫酸Cナトリウム(WAKO)及び50μg/mL ゲンタマイシンを添加したOpti-MEMTM I Reduced-Serum Medium, Liquid(Gibco社製)培地を40mL加えて,約16時間培養した。培養後,培地を回収して遠心(1500rpm,5分)し,得られた上清を0.2μmメンブランフィルターで濾過した。プレートに新しい培地を40mL加えて更に細胞を培養し,以後12〜24時間毎に培地の回収と新しい培地の添加を3〜5日間の間隔で繰り返して行った。回収した培地を遠心(1500rpm,5分)し,得られた上清を0.2μmメンブランフィルターで濾過した。濾過後の培地をヒト間葉系幹細胞の馴化培地(hMSC馴化培地)とした。hMSC馴化培地は,4℃又は凍結(-30℃)して保存し,4℃で保存する場合は,保存期限を10日間とした。このようにして調製したhMSC馴化培地は,以下に詳述するヒト角膜内皮細胞の初代培養及び継代培養において,ヒト角膜内皮細胞培養用基本培地に代えて,そのまま又は他の培地に添加して,ヒト角膜内皮細胞培養用の培地として使用することができる。
研究用ヒト角膜組織(シアトルアイバンク社製)から,鑷子を用いてヒト角膜内皮細胞を基底膜(デスメ膜)とともに剥離させた。剥離させたヒト角膜内皮細胞に,コラゲナーゼ溶液(コラゲナーゼを1mg/mLの濃度で添加したOpti-MEMTM I Reduced-Serum Medium)を添加して静置し,基底膜よりヒト角膜内皮細胞を剥離させた。次いで細胞を軽く振り混ぜて懸濁させ,これにヒト角膜内皮細胞培養用基本培地(8% FCS,200μg/mL CaCl2・2H2O,0.08%(w/v) コンドロイチン硫酸Cナトリウム(WAKO), 20μg/mL アスコルビン酸,50μg/mL ゲンタマイシン,5ng/mL EGF,1μM SB431542(TOCRIS社製)及び10μM Y27632(和光純薬工業)を添加したOpti-MEM I Reduced-Serum Medium, Liquid培地,以下「基本培地」)を添加して遠心(1200rpm,5分)し,細胞を回収した。細胞を,基本培地で懸濁し,細胞培養用プレート(コーニング社製)に,フラスコの底面の3分の1が細胞で占められる密度となるように播種し,37℃,5%CO2存在下で培養した。初代培養は,細胞がコンフルエントに達するまで行い,この間,培地を2日毎に交換した。
上記初代培養で,コンフルエントになるまで培養したヒト角膜内皮細胞を,培地を除去してPBS(-)で2回洗浄した後,トリプシン処理して剥離した。基本培地を加えて細胞を懸濁させた後,遠心して細胞を回収した。細胞を,基本培地で懸濁させて,細胞培養用プレートに,プレート底面の約3分の1が細胞で占められるように播種し,コンフルエントになるまで培養した。この間,培地を2日毎に交換した。
細胞培養用プレートから培地を除去し,PBS(-)で細胞を1回洗浄した後,細胞培養用プレートにPBS(-)を添加し,5%CO2存在下,37℃で10分間,細胞を静置させた。PBS(-)を除去し,TrypLETM select 10×(Life Technologies)を添加し,5%CO2存在下,37℃で10分間,細胞を静置させた。次いで,PBS(-)を添加し,細胞が単一の細胞にまで分離するようにピペッティングしてから,細胞を遠沈管に回収した。遠心(1200 rpm,5分)して細胞を沈殿させて上清を除去し,次いで基本培地を添加して細胞を懸濁させ,その一部を分取して生細胞数を計測した。
上記6種類の細胞保存試液中で4℃で72時間静置した後のヒト角膜内皮細胞の生存率は,(i):セリオキープで82.5%,(ii):25μg/mLのエピガロカテキンガレートを含有するセリオキープで96.7%,(iii):10μM Y27632を含むOpti-MEM I Reduced-Serum Medium, Liquidで5.1%,(iv):25μg/mLのエピガロカテキンガレートと10μM Y27632とを含むOpti-MEMTM I Reduced-Serum Medium, Liquidで67.3%,(v):オペガードで0%,及び(vi):25μg/mLのエピガロカテキンガレートを含有するオペガードで23.7%,であった。以上の結果は,25μg/mLのエピガロカテキンガレートを含有するセリオキープが,懸濁状態のヒト角膜内皮細胞の死滅を防止するうえで最も効果的であることを示すものであり,(ii)のエピガロカテキンガレートを含有する溶液中にヒト角膜内皮細胞を含んでなる組成物が,(i)〜(vi)の中で,角膜内皮機能不全患者への移植用組成物として最も好適であることを示すものである。また,仮に移植用の細胞に求められる好適な細胞の生存率を95%以上と想定すると,(ii)の細胞保存試液中での72時間経過後の細胞の生存率が96.7%であることから,(ii)の細胞保存試液を用いて調製した組成物に含まれるヒト角膜内皮細胞は,調製後少なくとも72時間は,角膜内皮機能不全患者への移植用に使用できる。
Claims (2)
- エピガロカテキンガレートを含有する溶液中にヒト角膜内皮細胞を含んでなる, 角膜内皮機能不全患者にヒト角膜内皮細胞を移植するために調製された,組成物であって、該エピガロカテキンガレートの濃度が15〜35μg/mLである、組成物。
- 該エピガロカテキンガレートの濃度が25μg/mLである,請求項1に記載の組成物。
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