JP4745386B2 - 組織増加のための分化した未成熟脂肪細胞および生分解性骨格の移植 - Google Patents
組織増加のための分化した未成熟脂肪細胞および生分解性骨格の移植 Download PDFInfo
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Description
S Eremia et al.,2000, Dermatol. Sur. 26, 1150-1158 Fulton JE et al.,2001, Dermatol.Clin.19(3), 523-530 G Sattler et al.,2000,Dermatol. Sur. 26, 1140-1144 JM Gimble et al.,2000, Bone 19:421-428 K Toriyama et al., 2002,Tissue Engineering, vol.8, 157-165 CW Patrick et al.,2002,Tissue Engineering, vol.8,283-293
(a)自家または同種脂肪組職から分離した未分化脂肪前駆細胞を脂肪細胞に分化させる段階;
(b)直径20〜40μmの分化した未成熟脂肪細胞を収集する段階;次いで
(c)収集された未成熟脂肪細胞と生分解性骨格をインビトロで一緒に培養する段階
を含む、移植用脂肪細胞組成物の製造方法を提供する。
(a)自家または同種脂肪組職から分離した未分化脂肪前駆細胞を脂肪細胞に分化させる段階;
(b)直径20〜40μmの分化した未成熟脂肪細胞を収集する段階;
(c)収集された未成熟脂肪細胞と生分解性骨格をインビトロで一緒に培養する段階;次いで
(d)未成熟脂肪細胞と生分解性骨格を一緒に体内に移植する段階
を含む、脂肪細胞移植方法を提供する。
(発明の実施するための最良の形態)
本明細書において、「脂肪前駆細胞(preadipocyte)」は脂肪組職から分離された細胞で、特に脂肪細胞に分化され得る可能性を有する細胞を意味する。
(1)脂肪吸入で得られた脂肪組職から脂肪前駆細胞の分離
脂肪組職をKRB溶液で洗浄し脂肪組職をコラゲナーゼで処理した後遠心分離して上層の脂肪階を除去し、下層に基質培地(stromal medium)を入れて懸濁させた後に遠心分離して下層の脂肪前駆細胞を回収する。
(2)脂肪前駆細胞を基質培地で培養
脂肪前駆細胞を基質培地に懸濁させて培養容器に加えた後、培養して底に付ける。基質培地は10%ウシ胎児血清が含まれているDMEM/F12(Dulbecco's Modified Eagle Medium/Ham's F12 Nutrient Broth)培地で、24時間培養する。
(3)増殖培地(expansion media)で培養
基質培地を除去した後、増殖培地で培養して十分な量の脂肪前駆細胞で増殖させる。増殖培地は10%ウシ胎児血清、EGF(epidermal growth factor)、bFGF(basic fibroblast growth factor)、TGF−beta 1(transforming growth factor beta 1)を含むDMEM/F12で、脂肪前駆細胞を迅速に増殖させて細胞量を大量に増加させる作用をする。
細胞が培養容器の底に単一層で満杯になると増殖培地を除去し基質培地を入れて1〜3日間追加培養する。
基質培地を除去し分化培地で培養する。分化培地は3%ウシ胎児血清、 ビオチン、パントテナート(pantothenate)、インスリン、デキサメダゾン、イソブチルメチルキサンチン(IBMX)、インドメタシンなどが含まれたDMEM/F12であって、脂肪前駆細胞が脂肪細胞に分化されるよう誘導し、この時細胞質に小さな脂質滴が形成され始める。
分化培地を除去し分化維持培地で培養する。分化維持培養は分化培地で細胞分化を誘導するイソブチルメチルキサンチンとインドメタシンを除外したもので、2〜3日毎に新しく取り替える。細胞は維持培地で脂質滴の数がより増えて大きさが次第に大きくなり脂肪細胞に成熟する。
特に、初期脂肪組織から脂肪前駆細胞を分離して培養容器に分株するとき、細胞濃度は、脂肪吸入によって得られた脂肪の場合、約0.04ml/cm3の脂質濃度とし、6〜8日培養して十分に増殖させる。継代培養なしにパッセージ0(P0)から増殖過程を終えて分化させることにより分化率が極大化された脂肪細胞を得ることができる。
(1)分化した未成熟脂肪細胞の収集
分化維持培地で培養される脂肪細胞は2〜3日間隔で新しい培地に交換しながら維持する。6〜9日間培養された脂肪細胞をトリプシン/EDTAを利用して収集し細胞の大きさを測定する。特に、収集された細胞を人に適用する場合、細胞収集2〜4日前の培養液からはウシ胎児血清を含まないことが望ましい。
(2)生分解性骨格
生分解性骨格の材料としてはフィブリン、アルギネート(alginate)、PLGA(poly lactic-co-glycolic acid)、PTFT(polytetrafluoroethylene)などが含まれるが、これに限定されない。その形態もまた注射として注入可能なものと、構造物として外科的手術を通して移植可能なものの全てを含む。
(3)分化した未成熟脂肪細胞を生分解性骨格に移植
直径20〜40μmの未成熟脂肪細胞を生分解性骨格に移植し培養培地で培養する。
(4)生分解性骨格内脂肪細胞の結合及び成熟を測定
生分解性骨格内で脂肪細胞の生着と脂肪細胞の成熟は、
(i)オイルレッドO染色を実施して脂肪細胞を同定し、脂肪細胞の成熟及び大きさを測定する方法;または
(ii)細胞培養上清液のレプチンを定量して脂肪細胞の機能を確認する方法で行われる。
まず脂肪吸入によって得られた脂肪組織から次のように脂肪前駆細胞を分離した。:血液を除去するために脂肪組織を同じ体積のKRB溶液に3〜4回洗浄した。脂肪組織と同じ体積のコラゲナーゼ溶液を入れて37℃の水浴で反応させた。これを遠心分離用チューブに移し入れて20℃、1200rpmで10分間遠心分離した。上層液のリピッドと脂肪層を除去し、下層のコラーゲン溶液を揺らさないように注意して分離した。基質培地を入れて懸濁させた後、20℃、1200rpmで5分間遠心分離した。この時、下に沈むものが脂肪前駆細胞であり、上層液を除去した。
(1)分化した未成熟脂肪細胞の収集
実施例1で生産された未成熟脂肪細胞を含んでいるプラスチックにトリプシン/EDTA溶液を添加し1〜15分間インキュベーターで反応させた後DMEM/F12を入れてトリプシン/EDTA溶液を不活性化させた。
フィブリノーゲン溶液とトロビン溶液は使用10分前に次の通り準備した。:アプロチニン溶液を凍結乾燥されたフィブリノーゲンが入っているバイアルに入れ1〜2分放置した後、軽く振って完全に溶かした。トロンビン溶液は塩化カルシウム溶液をトロビンが入っているバイアルに入れて内容物が完全に溶けるまで振って作った。
アルギネートはPBSに溶かして2%溶液に作った後、滅菌し室温保管した。塩化カルシウムと塩化ナトリウム溶液は各々102mM、150mMに作り滅菌した後室温保管した。
フィブリノーゲン溶液とトロビン溶液は使用する10分前に次のように準備した。アプロチニン溶液を凍結乾燥したフィブリノーゲンが入っているバイアルに入れ1〜2分放置し軽く振って完全に溶かした後、注射器に充填させた。トロビン溶液は塩化カルシウム溶液をトロビンが入っているバイアルに入れて内容物が完全に溶けるまで振って作った。
(産業上の利用分野)
Claims (5)
- 脂肪組織由来の脂肪前駆細胞から脂肪細胞に分化させて得られる直径20〜40μmの未成熟脂肪細胞を含む、身体体積代替のための移植用脂肪細胞組成物。
- 哺乳類の自家移植または同種移植に使用される請求項1に記載の組成物。
- 前記哺乳類がヒトである請求項2に記載の組成物。
- (a)自家または同種脂肪組織から分離された未分化脂肪前駆細胞を脂肪細胞に分化させる段階;
(b)直径20〜40μmの分化した未成熟脂肪細胞を収集する段階;次いで
(c)収集された未成熟脂肪細胞と生分解性骨格をインビトロで一緒に培養する段階
を含む、移植用脂肪細胞組成物の製造方法。 - (d)生分解性骨格内で脂肪細胞の結合及び成熟を測定する段階がさらに含まれる請求項4に記載の方法。
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KR1020050033091A KR100907248B1 (ko) | 2005-04-21 | 2005-04-21 | 분화된 어린 지방 세포와 생분해성 중합체의 이식에 의한신체의 부피 대체 방법 |
KR10-2005-0033091 | 2005-04-21 | ||
PCT/KR2006/001516 WO2006112684A1 (en) | 2005-04-21 | 2006-04-21 | Transplantation of differentiated immature adipocytes and biodegradable scaffold for tissue augmentation |
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KR102473820B1 (ko) * | 2019-03-21 | 2022-12-05 | (주)안트로젠 | 중간엽줄기세포-하이드로겔을 함유하는 주사형 조성물 및 이의 제조, 동결 및 해동방법 |
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US6153432A (en) * | 1999-01-29 | 2000-11-28 | Zen-Bio, Inc | Methods for the differentiation of human preadipocytes into adipocytes |
US20030161816A1 (en) * | 2001-12-07 | 2003-08-28 | Fraser John K. | Systems and methods for treating patients with processed lipoaspirate cells |
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TWI348372B (en) | 2011-09-11 |
KR100907248B1 (ko) | 2009-07-10 |
KR20060110637A (ko) | 2006-10-25 |
ATE513902T1 (de) | 2011-07-15 |
JP2009500048A (ja) | 2009-01-08 |
TW200801186A (en) | 2008-01-01 |
EP1874921A1 (en) | 2008-01-09 |
CN101203601B (zh) | 2010-12-08 |
US20080286241A1 (en) | 2008-11-20 |
EP1874921B1 (en) | 2011-06-22 |
CN101203601A (zh) | 2008-06-18 |
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