JP5271076B2 - 新規な細胞組成物およびその調製方法 - Google Patents
新規な細胞組成物およびその調製方法 Download PDFInfo
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- JP5271076B2 JP5271076B2 JP2008507685A JP2008507685A JP5271076B2 JP 5271076 B2 JP5271076 B2 JP 5271076B2 JP 2008507685 A JP2008507685 A JP 2008507685A JP 2008507685 A JP2008507685 A JP 2008507685A JP 5271076 B2 JP5271076 B2 JP 5271076B2
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Description
本出願は、米国特許出願第11/110,879号(2005年4月21日出願)の継続出願である(この出願は、引用によりその全体が本明細書に組み込まれる)。
(A)凍結して融解した肝細胞を密度勾配分画(特に、パーコール密度遠心分離)に付し、生存肝細胞を非生存肝細胞から分離し、
(B)分離した生存肝細胞を取得し、および
(C)取得した生存肝細胞を凍結保存し、それにより所望の肝細胞調製物を形成すること、
を含む。
本発明で使用する一次肝細胞の単離を可能とするために、多種多様な任意の方法を使用しまたは適合させ得る。たとえば、肝細胞の単離に好適な手法は、Morsianiら,(1995)“Automated Liver Cell Processing Facilitates Large Scale Isolation And Purification Of Porcine Hepatocytes”,ASAIO Journal 41:155−161およびSeglen,P.O.(1976)“Preparation Of Isolated Rat Liver Cells”,Meth.Cell Biol.13:29−83)に概要が示されている。Li,A.P.ら(1992)“Isolation And Culturing Of Hepatocytes From Human Liver”,J.Tissue Cult.Meth.14:139−146に記載された2ステップのコラゲナーゼ消化手順に対しては、具体的な言及がなされている。
本発明の肝細胞は、好ましくは液体窒素を用いて、最も好ましくはその単離から36時間以内に凍結保存する。ヒト肝細胞の凍結保存に関する考慮事項は、Lloyd,T.D.R.ら(2003)Cryopreservation Of Hepatocytes:A Review Of Current Methods For Banking”,Cell and Tissue Culture Banking 4:3−15で考察されている。また、肝細胞を凍結保存するための好適な手順については、下記の文献:Adams,R.M.ら(1995)“Effective Cryopreservation And Long−Term Storage Of Primary Human Hepatocytes With Recovery Of Viability,Differentiation,And Replicative Potential”,Cell Transplant.4(6):579−586;Chesne,C.ら(1993)“Viability And Function In Primary Culture Of Adult Hepatocytes From Various Animal Species And Human Beings After Cryopreservation”,Hepatology 18(2):406−414;Coundouris,J.A.ら(1993)“Cryopreservation Of Human Adult Hepatocytes For Use In Drug Metabolism And Toxicity Studies”,Xenobiotica.23(12):1399−1409;Hewitt,N.J.ら(2004)Cryopreserved Rat,Dog And Monkey Hepatocytes:Measurement Of Drug Metabolizing Enzymes In Suspensions And Cultures”,Hum Exp Toxicol.23(6):307−316;Novicki,D.L.ら(1982)“Cryopreservation Of Isolated Rat Hepatocytes”,In Vitro.18(4):393−399;Shaddock,J,G.ら(1993)“Cryopreservation And Long−Term Storage Of Primary Rat Hepatocytes:Effects On Substrate−Specific Cytochrome P450−Dependent Activities And Unscheduled DNA Synthesis”,Cell Biol Toxicol.9(4):345−357;Zaleski,J.ら(1993)“Preservation Of The Rate And Profile Of Xenobiotic Metabolism In Rat Hepatocytes Stored In Liquid Nitrogen”,Biochem Pharmacol.46(1):111−116にも見られる。
凍結試料は、さらなる処理のために、液体窒素または液体窒素蒸気の存在下から取り出すことにより融解され得る。凍結試料は、好ましくは、予備加温した約37℃〜約42℃の水浴に直ちに入れることにより融解する。好ましくは、少なくとも試料容器を逆さにしたときに氷の塊が外れ得る段階まで、細胞を融解する。融解した細胞は、次いで好ましくは急速に処理し、たとえばパーコール勾配遠心分離(後述)あるいは逐次洗浄によって、DMSOとの接触から取り除く。
本発明の一態様は、融解した細胞を、後に1回以上再凍結および再融解され得るように再構築する能力に関する。こうした反復凍結保存肝細胞調製物は、多くの用途を有する。該調整物は、バイオ人工肝臓、肝細胞移植片、肝臓補助デバイス、肝細胞移植、およびインビトロの用途において使用され得る。特に、反復凍結保存肝細胞調製物は、インビトロ薬物代謝研究(たとえば、特有の性質(たとえば、代謝性多型、遺伝的多型など)を有する肝細胞の同定)、生体異物の代謝的運命に関する研究および肝細胞の薬物代謝性酵素プロフィールの改変における生体異物の影響に関する研究、肝臓の酵素および機能(たとえば、コレステロール代謝)に対する生体異物のIC50を決定するための阻害研究、生体異物を用いた遺伝子導入研究、生体異物を用いたタンパク質導入研究、肝細胞に対する生体異物の毒性評価、生体異物を用いた輸送研究(たとえば、P−糖タンパク質輸送系、有機イオン輸送体、有機カチオン輸送体などに関する研究)、生体異物を用いた代謝性クリアランス研究、ならびに有効性アッセイ(たとえば、リポタンパク質プロセシング、糖新生、タンパク質分泌など)において使用され得る。反復凍結保存肝細胞調製物はまた、肝炎ウイルスならびに他の感染性ウイルスおよび病原体を研究あるいは増殖させるために使用され得る。取得した細胞は、DNA、mRNAもしくはプロテオミクス研究における使用のため、または代謝性多型の研究のために再構築され得る。反復凍結保存肝細胞調製物はまた、代謝性クリアランス研究および有効性アッセイ(たとえば、リポタンパク質プロセッシング、糖新生、タンパク質分泌など)において使用され得る。細胞は、大規模インキュベーションのためのバイオリアクターへの播種における使用、もしくはミクロRNAによる遺伝子調節モデルとしての使用、または他の細胞型(たとえば、肝臓由来の非実質細胞もしくは他の供給源由来の細胞、たとえばCaco−2細胞)との組み合わせ系における使用のために再構築され得る。
肝細胞の凍結および融解を繰り返すことのできる本発明の能力によって、プールした肝細胞調製物、特にプールしたヒト肝細胞調製物の作製がさらに容易になる。上記したように、個々の肝臓試料から得られる肝細胞は、異なる代謝能を有している。肝細胞の再生可能な使用または研究を促進するため、種々の供給源に由来する肝細胞をプールして複合すなわち「平均的」な肝細胞調製物を得ることにより、こうした試料のばらつきに起因する肝細胞間の差異を最小限にすることが望ましい。したがって、こうした複合肝細胞調製物は、「平均的」な肝細胞試料の代謝活性を有する調製物、または新たに単離された肝細胞の肝細胞酵素機能に近い肝細胞酵素機能を有する調製物を提供するように構成され得る。こうした代謝活性としては、たとえば、下記の酵素活性の一部および全部を含む:クマリン7−ヒドロキシラーゼ(COUM)、デキストロメトルファンO−脱メチル化酵素(DEX)、7−エトキシクマリンO−脱エチル化酵素(ECOD)、7−ヒドロキシクマリン(7−HCG)の第2相代謝に関与する活性、7−ヒドロキシクマリン(7−HCS)の第2相代謝に関与する活性、メフェニトイン4−ヒドロキシラーゼ(MEPH)、テストステロン6(β)−ヒドロキシラーゼ(TEST)、トルブタミド4−ヒドロキシラーゼ(TOLB)、フェナセチンO−脱エチル化酵素(PHEN)、またはクロルゾキサゾン6−ヒドロキシラーゼ(CZX)。こうした代謝活性アッセイを行うための基質、測定方法およびアッセイ単位を表1に示す。
凍結保存肝細胞は、以下に示すようにして融解し、再凍結させる。
1) 循環式水浴加熱器を37〜42℃に設定する。
2) およそ200〜400mlのInvitroGro CP−2培地を1〜2Lのビーカーに加える。生物学的安全キャビネットに手動ピペットまたは液体操作ロボットを取り付ける。
3) およそ50個のクリオバイアルをデューアー容器から取り出し、2つの試験管ラックに速やかに配置する。可能な場合は、バイアルの間隔をあける。
4) 固体の細胞懸濁液を加熱水浴中に浸し、バイアルを逆さにしたときに氷の塊が外れる状態になるまで浸しておく。
5) 細胞懸濁液を各バイアルからビーカー内に注入する。1mlのInvitroGro CP−2培地を該小ビーカーから各バイアル内に加えてすすぎ、内容物を該ビーカー内に注入する。融解した細胞懸濁液を1Lの滅菌バッグに移す。
6) バッグ、すなわち2Lバッグ中のInvitroGro CP−2培地および30%の割合でバッグに入れたパーコールを、COBE自動細胞処理装置に取り付け、標準的手法従って処理する。
7) 細胞の計数を行う。
8) 細胞懸濁液を凍結保存する。
個々の(プールしていない)肝細胞の種々の供給源の試料間のばらつきを示すため、肝細胞を82例の異なるドナーから単離し、細胞生存率および酵素機能について分析する。下記の代謝活性:COUM、DEX、ECOD、7−HCG、7−HCS、MEPH、TEST、TOLB、PHEN、およびCZXを評価する。結果を表3に示す。
肝細胞の凍結保存プールロットを調製し、融解後の生存率および酵素機能について解析する。以下の代謝活性:COUM、DEX、ECOD、7−HCG、7−HCS、MEPH、TEST、TOLB、PHEN、およびCZXを評価する。
凍結保存肝細胞の一般的な使用は、該肝細胞を融解し、次いでこれを種々の生体異物とともにインキュベートすることである。この目的のため、少なくとも数時間、肝細胞がその生存率を維持することが好ましい。プールした凍結保存肝細胞の1ロットについて融解後生存率の経時変化を調べるため、細胞を融解し、12ウェルプレートのウェルに分注し、5%CO2中37℃でインキュベートした。次いで、肝細胞の生存率を複数の時点で6時間まで測定する。表6はこの解析結果を示し、6時間の時点で肝細胞の39%が生存状態を維持していた。
Claims (9)
- 肝細胞を少なくとも2回凍結して融解することが可能であり、調製物の肝細胞のうち70%を超える細胞が最後の融解後に生存している、反復凍結保存肝細胞の所望の調製物を製造する方法であって、
(A)凍結して融解した肝細胞を密度勾配分画に付し、生存肝細胞を非生存肝細胞から分離し、
(B)分離した生存肝細胞を取得し、および
(C)取得した生存肝細胞を凍結保存し、それにより前記所望の肝細胞調製物を形成し、2回目に肝細胞を融解した後に密度勾配分画を用いず、1回目と2回目の凍結保存の間に肝細胞をプレーティングせず、前記所望の肝細胞の調製物の肝細胞のうち70%を超える細胞が最後の融解後に生存していることを特徴とする反復凍結保存肝細胞調製物の製造方法。 - 前記密度勾配分画が、ポリビニルピロリドンでコートされたコロイド状シリカ粒子による密度分離を含む、請求項1に記載の方法。
- 前記肝細胞が、ヒト肝細胞、ブタ肝細胞、サル肝細胞、イヌ肝細胞、ネコ肝細胞、ウシ肝細胞、ウマ肝細胞、ヒツジ肝細胞、および齧歯類肝細胞からなる群より選択される、請求項1に記載の方法。
- 前記肝細胞がヒト肝細胞である、請求項3に記載の方法。
- 前記調製物が、複数の供給源の肝細胞をプールした調製物を含む、請求項1に記載の方法。
- 前記複数の供給源が同一の性別、種または健康状態のものである、請求項5に記載の方法。
- 前記プールした肝細胞調製物の肝細胞が前記プールした調製物に所望のレベルの代謝活性をもたらす、請求項5に記載の方法。
- 前記代謝活性が、COUM、DEX、ECOD、7−HCG、7−HCS、MEPH、TEST、PHEN、およびCZXからなる群より選択される、請求項7に記載の方法。
- 前記調製物の肝細胞の80%を超える細胞が生存している、請求項1に記載の方法。
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CN109055305B (zh) * | 2018-09-17 | 2021-03-26 | 河南科技大学 | 一种奶牛肝干细胞的分离提取方法 |
WO2024026313A1 (en) | 2022-07-25 | 2024-02-01 | The Regents Of The University Of California | Methods of producing and using avian embryonic stem cells and avian telencephalic organoids |
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ATE165208T1 (de) | 1990-01-17 | 1998-05-15 | Univ California | Zusammensetzung zur verbesserung der lebenserhaltung biologischer materialien |
US5795711A (en) | 1996-04-04 | 1998-08-18 | Circe Biomedical, Inc. | Cryopreserved hepatocytes and high viability and metabolic activity |
US5895745A (en) | 1996-09-25 | 1999-04-20 | W.R. Grace & Co.-Conn. | Method of thawing cryopreserved cells |
ATE373079T1 (de) | 1999-06-21 | 2007-09-15 | Gen Hospital Corp | Zellkultursysteme und verfahren für einrichtungen zur organunterstützung |
HUP0203781A2 (hu) | 2000-01-19 | 2003-03-28 | University Of North Carolina At Chapel Hill | Májszövet-forrás |
AU2001260658A1 (en) | 2000-06-01 | 2001-12-11 | Hokkaido Technology Licensing Office Co., Ltd. | Method of preparing small hepatocytes preservable in freeze-dried state and method of preserving the same in freeze-dried state |
WO2003105663A2 (en) | 2002-06-01 | 2003-12-24 | Stelsys Llc | Liver assist system based on hollow fiber cartridges or rotating bioreactor |
EP1576117A4 (en) | 2002-07-19 | 2006-02-01 | Vesta Therapeutics Inc | METHOD FOR OBTAINING VIABLE HUMAN LIVER CELLS, INCLUDING STEM CELLS / HEPATIC PRECURSORS |
US20040265997A1 (en) | 2003-06-27 | 2004-12-30 | Park Sung-Soo | Bio-artificial liver system |
US7604929B2 (en) | 2005-04-21 | 2009-10-20 | In Vitro Technologies, Inc. | Cellular compositions and methods for their preparation |
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CA2604693A1 (en) | 2006-11-02 |
CN101501186A (zh) | 2009-08-05 |
AU2006240454A1 (en) | 2006-11-02 |
WO2006115682A3 (en) | 2009-04-23 |
CN101501186B (zh) | 2013-06-19 |
PL1891208T3 (pl) | 2012-11-30 |
PT1891208E (pt) | 2012-06-20 |
EP1891208B1 (en) | 2012-06-06 |
FI2479259T3 (fi) | 2024-02-20 |
ES2965619T3 (es) | 2024-04-16 |
US20050239042A1 (en) | 2005-10-27 |
US20100075294A1 (en) | 2010-03-25 |
CN103275924A (zh) | 2013-09-04 |
EP2479259A2 (en) | 2012-07-25 |
US7604929B2 (en) | 2009-10-20 |
AU2006240454B2 (en) | 2010-07-22 |
CA2604693C (en) | 2012-08-14 |
US20100075295A1 (en) | 2010-03-25 |
AU2010235914A1 (en) | 2010-11-11 |
LT2479259T (lt) | 2024-03-12 |
EP2479259A3 (en) | 2012-08-01 |
HK1113689A1 (en) | 2008-10-10 |
JP2008539697A (ja) | 2008-11-20 |
ES2389099T3 (es) | 2012-10-23 |
EP2479259B1 (en) | 2023-11-22 |
EP1891208A2 (en) | 2008-02-27 |
DK2479259T3 (da) | 2024-02-19 |
EP2218777A1 (en) | 2010-08-18 |
DK1891208T3 (da) | 2012-07-16 |
WO2006115682A2 (en) | 2006-11-02 |
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