JP2021518118A - 神経幹細胞の高効率分離培養方法 - Google Patents
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Abstract
Description
ヒトの脳組織の重量を測定した後、2〜3回PBSを使用してウォッシングを行った。組織を適切なサイズに切り取った後、エンザイム溶液(10 units/ml of papain、0.1mg/ml of DNase I、4mg/ml of D、L−cysteine)に入れた後、ブレードと手術用はさみを使用して、物理的に小片に切り取った。37℃で30分間処理後、使い捨てピペット(disposable pipette)を使用して組織を細かく破砕した後、70uMメッシュを使用してろ過した後、PBS(Phosphate Buffered Saline)を使用してウォッシングを行った。
神経幹細胞を70〜80%程度のコンフルエンシーで培養した後、PBSで2回ウォッシングした。神経分化条件であるDMEM/F12、0.5%FBS、B27、0.5mM IBMX、P/Sを処理した後、48時間後に細胞を固定した後、免疫染色を行った。
神経幹細胞は、PLOがコーティングされた8 well chamber slide(thermo)に培養した後、4%パラホルムアルデヒド(paraformaldehyde)に5分間固定させた。0.1%Triton−X100が希釈されているPBS(0.1%PBST)で水洗した後、ブロッキング(blocking)溶液(5%normal goat serum、5%normal donkey serum)で1時間程度処理した後、1次抗体を処理して一晩の間(overnight)反応させ、翌日、PBSで2回ウォッシング後、Alexa488またはAlexa594が結合された2次抗体を1時間の間処理した。DAPIでカウンターステイニング(counter staining)を行った後、マウンティング後に蛍光顕微鏡で観察した。
クランプ培養方法の最適条件を探すために、ヒトの脳組織にエンザイム溶液を処理し、物理的に細かく分割した後、様々なサイズのメッシュ(mesh)にかけてサイズ別にクランプを獲得した(図4a参照)。クランプをサイズ別に下記のようにタイプI〜タイプIVを決めて、その形態を顕微鏡で観察した。
70μm<クランプタイプII<100μm
40μm<クランプタイプIII<70μm
クランプタイプIV<40μm
クランプのサイズ別分離培養効果を比較するため、前記実施例1の方法によって2名の患者から由来したクランプタイプI〜IVを培養してコロニーの数字を確認し、顕微鏡でコロニーの形状などを観察した。
本発明による方法でクランプタイプIIを培養した場合とパーコールを用いた単一細胞培養時の効果を比較するため、前記実施例1の方法によって細胞を培養し、培養成長曲線を作成し、クランプタイプIIの継代培養による細胞の形態を顕微鏡で観察した。
臍帯由来の血管内皮細胞は、Promocellから購入し、供給者の専用培地を使用した。200μLのPhenol red−free Matrigel(BD)に1x106血管内皮細胞と1x106神経幹細胞を混ぜた後、4−6週齢のBalbc−nuマウスの皮下に移植した。3−4日後にMatrigel除去した後、4%PFAで24時間の間固定した後、ブロックを製作した。組織学的分析のためにヘマトキシリン&エオジン(H&E)染色を行い、Matrigel内に生成された新生血管を染色するため、血管内皮細胞と神経幹細胞を区別するためにCD31とalpha−smooth muscle actin(α−SMA)で免疫蛍光染色を行った。
Claims (5)
- 脳組織を酵素溶液に入れて酵素処理する段階と、
酵素処理された脳組織から物理的に細胞クランプ(clump)を分離する段階と、
前記細胞クランプをサイズに応じて分離し、不純物を除去する段階と、
前記細胞クランプを培養皿に接種して継代培養する段階と、
を含む、神経幹細胞の高効率分離培養方法。 - 前記酵素溶液は、パパイン(papain)と、DNase Iと、D、L−システイン(cysteine)と、を含む、請求項1に記載の神経幹細胞の高効率分離培養方法。
- 前記細胞クランプは、70〜100umサイズを有するものである、請求項1に記載の神経幹細胞の高効率分離培養方法。
- 前記脳組織は、成人の脳組織である、請求項1に記載の神経幹細胞の高効率分離培養方法。
- 前記神経幹細胞の分離培養方法は、パーコール(percoll)を用いた単一細胞分離段階を含んでいないことを特徴とする、請求項1に記載の神経幹細胞の高効率分離培養方法。
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KR10-2018-0031101 | 2018-03-16 | ||
KR20180031101 | 2018-03-16 | ||
KR10-2019-0011226 | 2019-01-29 | ||
KR1020190011226A KR101994640B1 (ko) | 2018-03-16 | 2019-01-29 | 신경줄기세포의 고효율 분리배양 방법 |
PCT/KR2019/001288 WO2019177270A1 (ko) | 2018-03-16 | 2019-01-30 | 신경줄기세포의 고효율 분리배양 방법 |
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EP1827108B1 (en) * | 2004-11-29 | 2015-04-08 | Yeda Research And Development Co., Ltd. | Induction of neurogenesis and stem cell therapy in combination with copolymer 1 |
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EXP. ANIM., vol. 51, no. 4, JPN6021043859, 2002, pages 383 - 390, ISSN: 0004636441 * |
NAT. PROTOC., vol. 6, no. 12, JPN6021043860, 2011, pages 1981 - 1989, ISSN: 0004636442 * |
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WO2019177270A1 (ko) | 2019-09-19 |
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EP3783099A1 (en) | 2021-02-24 |
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