我々は編集支援のためにJonnaエリスに感謝します。国立精神衛生研究所から：説明プロジェクトが受賞番号R01MH079751（F.ペルッツィPI）によってサポートされていました。内容はもっぱら著者の責任であり、必ずしも国立精神衛生研究所や国立衛生研究所の公式見解を示すものではありません。

1。ポリ-D-リジン（PDL）：準備<ol><li> 1 mg / mlのストック溶液を得るためにPDLの5mgに無菌のddH 2 Oの5ミリリットルを追加します。 </li><li>数回ピペッティングしてストック溶液を混ぜる。 </li><li>すぐに使用するか、または2-8ポリ-D-リジン溶液℃で保存してください。 </li></ol> 2。ポリ-D-リジン（PDL）：コーティングプラスチック細胞培養皿<ol><li>滅菌のddH 2 Oで10μg/ mlの最終濃度にPDLストック溶液を希釈します。 </li><li>培養面積（60 mmディッシュに3 ml）をカバーする60mmディッシュにピペット十分なソリューションを提供します。 </li><li>文化面にもコーティングを確保するために静かに揺する。 </li><li>一晩室温（RT）でコーティングしたプレートをインキュベートします。 </li><li>通常翌日に、解剖の日に、吸引によってポリ-D-リジン溶液を除去し、無菌のddH 2 Oを3mlで簡単に洗うこの手順を繰り返します。回目の洗浄後、吸引で完全に水を削除します。 </li><li>プレートは、最大3週間までは4℃で保存することができます。 </li></ol> 3。ポリ-D-リジン（PDL）およびラミニン：ガラスの作製とコーティング二チャンバースライド<ol><li>それぞれ10および5μg/ mlの最終濃度に無菌のddH 2 OのミックスPDL（1 mg / ml）とラミニン（1 mg / ml）のストック溶液を、。 </li><li>培養面積（2よくガラス二チャンバースライドの各ウェルに1 ml）をカバーするガラス二チャンバースライドの各ウェルにピペット十分なソリューションを提供します。 </li><li>文化面にもコーティングを確保するために静かに揺する。 </li><li>室温で一晩コーティングしたプレートをインキュベートします。 </li><li>次の日に、吸引を介してポリ-D-リジンラミニンコーティング溶液を除去し、無菌のddH 2 Oを1 mlで2回サッと洗う回目の洗浄後、吸引で完全に水を削除します。 </li><li>チャンバースライドは最大3週間までは4℃で保存することができます。 </li></ol>注：任意のガラスチャンバースライドは、コーティングされたfollowinすることができますグラムは、このプロトコル。各スライドは、コントロール·テスト実験の設定（例えば、未処理の対扱われ、非トランスフェクト対）を提供しますので、我々はしばしば二チャンバースライドを使用しています。 4。神経解剖と文化<ol><li> 37°Cの水浴中で以下の試薬を温める。 <ul type="disc"><li>元の100ミリリットルボトルのTrypLEエクスプレス。 </li><li> Neurobasal/B27完全培地（ 表Iを参照）。温めたボリュームは、（10 60ミリメートルメッキ皿例えば30ミリリットル）メッキになる料理の数によって異なります。 </li></ul></li><li> 4 60 mmの培養皿と15ミリリットルBDファルコン高透明ポリプロピレンコニカルチューブに13 mlの冷HibernateのE溶液3mlを追加します。 </li><li> 3 100 mmの培養ディッシュのそれぞれに冷たい解剖培地（ 表II、博士オリンピアメウッチ、個人的な通信を参照してください）の25〜30 mlを追加します。媒体の大容量を含むこれらのプレートは、すぐに彼らの後の胚を洗浄するために使用されます。羊水嚢（ステップ4.7および4.8）から除去。 </li><li>実験動物の人道的なケアと使用に関する公衆衛生サービスのポリシーに従って、制度承認された動物のケアの下でCO 2でE17タイミング妊娠ラットを安楽死させるとプロトコルを使用しています。 </li><li> 70％EtOHで下腹部をスプレーし、子宮および胚を露出はさみのペアを持つ皮膚や筋肉を介して内側にカット。 </li><li>すべての胎児を取り出して、冷たい解剖媒体の過剰を含む滅菌100-mmディッシュに置いてください（25-30ミリリットル、ステップ4.3を参照）。 </li><li>羊膜嚢からはさみの小さなペアを使用して胚を切断して冷たい解剖媒体を含む第二の100-mmディッシュに配置します。 </li><li> 5〜10秒間静かに傾けて、100 mmディッシュで室温で胚を洗浄します。その後、解剖媒体を含む第三100 mmディッシュにすすいだ胚を転送します。過剰培地で2回洗浄し、通常は血液の痕跡をすべて削除するのに十分である。ただし、NECessary、冷たい解剖培地の25〜30 mlを含有する新鮮な100-mmディッシュを使用して、1つより多くの時間を洗ってください。 </li><li>実体顕微鏡と湾曲した鉗子を用いて、皮膚や頭蓋骨を引き出して、それぞれのラット胚の脳を抽出します。氷の上でこれらのプレートを維持冷たいHibernateのE. 60-mmディッシュ（典型的には、皿当たりません5つ以上の脳を含む）のいずれかで、脳全体に配置します。 </li><li>一度に一皿を取ると、解剖顕微鏡下で、半球を分離し、中脳と髄膜を除去する大脳皮質を分離します。 </li><li>オプション：正中線に沿ってカットの脳は、海馬を抽出し、海馬ニューロンを分離するには、以下の手順に従ってください。 </li><li>すべての解剖が完了するまで氷上に大脳皮質を残す冷たいHibernateのE.の13ミリリットルを含む15 mlのコニカルチューブに明らかにあるすべての解剖皮質を収集します。その小さなサイズのために、解剖海馬ではなく、15 mlチューブに1.5 mlのエッペンドルフチュー​​ブに回収することができる。またはこのステップ野では、必要に応じて海馬は、2月4日皮質またはバイアルあたり2から4海馬の割合でHibernate E + 2％B27 +ゲンタマイシン（50μg/ ml）を+ファンギゾン（250 ng / ml）を1 mlを含むクライオチューブバイアルに配置することができます。脳組織は、最大一週間（後の時代はまだテストされていません）に、暗所で4℃で保存することができます。必要なときに、HibernateはEを含む15 mlチューブに脳組織を転送してから神経細胞を分離するには、以下のプロトコルに従うように微細な鉗子を使用しています。 </li><li>組織培養フードにチューブを転送します。皮質は、チューブの底に沈殿して、慎重に上清を除去することができます。 </li><li> 15 mlのコニカルチューブに新鮮なHibernateのEの13ミリリットルを追加して、野にはチューブの底に沈殿し、上清を注意深く除去することができます。このステップ2回以上繰り返すと、最後に洗浄した後、慎重にすべてのメディアを削除します。 </li><li>暖かいTrの、酵素1〜2ミリリットルを（海馬の分離のための以下を使用して野の数に応じて）を追加することによって大脳皮質を消化ypLEエクスプレス。パラフィルムでチューブのキャップを密封して10分間37℃の水浴中でチューブをフロートさせます。 </li><li>キャップを開ける前に、70％エタノールでチューブをスプレーし、Hibernate E. 10mlの皮質は、チューブの底に沈殿し、上澄みを除去するために許可を追加します。 TrypLE Expressを洗い流すには、この手順を3回繰り返します。最後のステップでは、慎重にすべてのメディアを削除します。 </li><li>穏やかに（4-5回）ファイアーポリッシュガラスパスツールピペット（直径約1mm）を使用してNeurobasal/B27完全培地2ml中の皮質をひいて粉にする。気泡がないように注意してください。 </li><li>直径の小さい滅菌ガラスパスツールピペット（すなわち直径1/2-3/4 mm程度ピペット）で別の4-5回を繰り返します。これより小さいパスツールピペットを使用していないか、それが細胞を破壊します。 </li><li>解決するために（もしあれば、一般的には非常に少ない）は、組織の残りの部分ができます。 </li><li>組織の定着部分を残して、新しい15 mlチューブに上部の単一細胞懸濁液を移す。毛皮Neurobasal/B27完全培地で10〜12ミリリットルに細胞懸濁液を希釈して配置する必要があります。 </li><li>よく混合し、1.5mlのエッペンドルフチューブ内で50倍カウント·ソリューション（ 表IIIを参照）490μlに細胞懸濁液10μlを添加することによりカウントするための細胞を希釈します。 </li><li> 5.0×10 4 / cm 2の密度で、PDLコーティングプレート上に細胞をプレートし。ヌクレオが実行されるならば、我々はより高い濃度（8-10×10 4個/ cm 2）で細胞をプレーティングをお勧めします。 </li><li>約13×10 6ニューロンがそれぞれE17胎児から導出されるように、通常、我々は、実験ごとに9月10日胎児を解剖。より多くの胚が必要な場合は、全体の手順は、2時間以上持続しないことを確認します。 </li><li> 24時間分離した後、必要に応じて、シトシン-β-D-アラビノフラノシド（AraCの）10μMのは、グリア細胞の増殖を防ぐために、それぞれの皿に追加することができます。 Neurobasal/B27媒体はグリア細胞の増殖を阻害するので、しかし、この手順は必須ではありません（Invitrogen社/ Gibco）を製造元の推奨に従って。 </li><li>正確な時間が所望の分化段階に依存するものの、神経細胞は、 試験管内で 4-5日後に実験に使用することができます。我々は、生存率の有意な減少（ 図1）を使用せずに最大4週間の培養神経細胞を持っています。 </li><li>拡張された培養については、新たに調製しNeurobasal/B27完全培地で毎週培養培地を交換してください。 </li></ol> 5。代表的な結果ガラスチャンバースライド上で培養したニューロンは、免疫細胞化学に付すことができる。 図1は、培養中の5日後に固定されており、神経突起を表示するには、抗MAP-2抗体で免疫標識皮質ニューロンの典型的なイメージを示しています。 図2は、文化の中で3週間後のラット海馬ニューロンの代表的なイメージを示しています。完全に分化した細胞のニューロンの形態は、MAP-2で強調表示されているImmunolabeling（MAP-2神経マーカー、マウスモノクローナル抗体クローンAP-20、ジーンテックス、カリフォルニア州アーバイン）は、以前1を説明されている標準的な手順に従って、。画像は、EXiアクアカメラ（Qimaging）を搭載したニコンのEclipse E400直立蛍光顕微鏡で可視化した電動Z軸、SlideBook5取得/デコンボリューションソフトウェア（インテリジェントイメージングイノベーションズ社、デンバー、コロラド州）。個々の画像の3次元画像のシリーズは、1つの2次元の画像にデコンボリューションと分解能を高めるために近くに最大の強度に信号のカットオフを調整することによって解決されました。 図3は、ニューロン培養の純度を示しています。タンパク質溶解物をDIV7ラット神経細胞培養（CTX）からとヒト神経膠芽腫（GBM）のケースから得られた。予想通り、神経細胞ライセートはアストロサイトのマーカーGFAPのために強く神経タンパク質MAP-2の正と負であり、GBMタンパク質溶解物が負のfである間、またはMAP-2およびGFAP陽性の。 私たちのプロトコルで我々がメディアを解剖し、洗浄など、いくつかの年のためにHibernate Eを使用してきましたが、最近我々は、さらに使用するために脳組織を維持するためにそれの追加と非常に実用的な使用を検討しています。 図4 は 、in vitro 5（DIV5） の日数を示しています胚からの元の郭清をした後に休止状態E + B27に1週間4°Cで保管皮質から単離されたラット皮質ニューロンの文化。ニューロンは、前述のようにPDLとラミニンでコーティングされたガラス2チャンバースライド上に播種した。取得した画像（ 図2）上記のようにSlideBook5取得/デコンボリューションソフトウェアを使用してデコンボリューションされました。 <img alt="図1" src="/files/ftp_upload/3965/3965fig1.jpg" /> 図1。pmaxGFP（Amaxa、ロンザ、ウォーカー、MD）でnucleofectedとMAPを免疫標識皮質ニューロンの代表画像-2抗体、赤インチオリジナルの倍率100倍。 <img alt="図2" src="/files/ftp_upload/3965/3965fig2.jpg" /> 図2。文化の中で3週間後の海馬ニューロンの、赤で、MAP-2免疫標識を示す代表的なイメージ。 DAPI染色は、青色で、細胞の核を示しています。オリジナルの倍率40倍。 <img alt="図3" src="/files/ftp_upload/3965/3965fig3.jpg" /> 図3：神経細胞培養の純度を示すウエスタンブロット。ラットニューロンおよびヒトGBMのタンパク質溶解物30μgを電気泳動により分離し、標準的な手順1を以下のウェスタンブロット解析に供した。抗MAP-2は細胞シグナリング（ダンヴァーズ、マサチューセッツ州）、抗GFAP抗体由来のポリクローナルウサギはChemicon社からのマウスモノクローナル抗体（ミリポア、ビルリカ、MA）であった、マウスモノクローナル抗GRB2抗体BDトランスダクションラボラトリーズからの実施であった（火花、メリーランド州）。 GRB2は、ローディングコントロールとして用いた。 </P&gt; <img alt="図4" src="/files/ftp_upload/3965/3965fig4.jpg" /> 図4代表vitroで5日間の写真（DIV5）に残された皮質から得られたラット大脳皮質ニューロンのHibernate Eその郭清後の一週間、4℃+ B27。 A）ガラス二チャンバースライド上で培養した神経細胞の位相コントラストを。オリジナルの倍率20倍。 B）免疫蛍光は緑色で、神経突起のMAP-2の発現を示す。文化はアストロサイトのマーカーGFAPに対して陰性であった。 DAPI染色は、青色で、細胞の核を示しています。オリジナルの倍率40倍。

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利害の衝突が宣言されません。

ここで説明したラット海馬および皮質ニューロンの解剖と文化の方法は化学的に定義された媒体（ 図3）で成長し、ほぼ純粋な神経細胞の培養物を用いて実験を行うことができます。無血清培地で培養し、ほぼ純粋なニューロンのプロトコルは以前に2,3,4を説明してきたが、我々の方法で行われた重要な変更があります。従来のプロトコルから別の（すなわち、バンカー?...

<table border="1"><tbody><tr><td> 試薬 </td><td> 濃度 </td></tr><tr><td> Neurobasal </td><td> 98パーセント</td></tr><tr><td> B27 </td><td>二パーセント</td></tr><tr><td>グルタミン</td><td> 0.5mMの</td></tr></tbody></table> 表I. Neurobasal/B27完全培地 。 <table border="1"><tbody><tr><td> 試薬 </td><td> 濃度 </td></tr><tr><td>グルコース</td><td> 16mMの</td></tr><tr><td>スクロース</td><td> 22 mmの</td></tr><tr><td> HEPES </td><td> 10mMの</td></tr><tr><td> NaClを</td><td> 160 mMの</td></tr><tr><td>塩化カリウム</td><td> 5mMの</td></tr><tr><td>のNa 2 HPO 4 </td><td> 1mMの</td></tr><tr><td> KH 2 PO 2 </td><td> 0.22 mMの</td></tr><tr><td>ゲンタマイシン</td><td> 50μg/ mlの</td></TR&gt; <tr><td>ファンギゾン</td><td> 250 ng / mlの</td></tr><tr><td> pHは</td><td> 7.4 </td></tr><tr><td>オスモル濃度</td><td> 320から330浸透圧</td></tr></tbody></table> 表II。解剖媒体 。 <table border="1"><tbody><tr><td> 試薬 </td><td> 量（μL） </td></tr><tr><td> Neurobasal/B27完全培地</td><td> 240 </td></tr><tr><td>トリパンブルーは0.4％の染色</td><td> 250 </td></tr><tr><td>合計</td><td> 490 </td></tr></tbody></table> 表III。ソリューションを数える50倍 。 <table border="1"><tbody><tr><td> 試薬 </td><td> 会社 </td><td> 猫。数 </td></tr><tr><td> HibernateはE </td><td> Brainbits </td><td> 767171 </td></tr><tr><td> Neurobasal </td><td> Gibco社、Invitrogen社</td><td> 21103-049 </td></tr><tr><td> B27 </td><td> Gibco社、Invitrogen社</td><td> 17504-044 </td></tr><tr><td>ファンギゾン</td><td> Gibco社、Invitrogen社</td><td> 15290-018 </td></tr><tr><td>硫酸ゲンタマイシン</td><td>シグマアルドリッチ</td><td> G1264 </td></tr><tr><td>グルタミン200mMの</td><td> Gibco社、Invitrogen社</td><td> 35050 </td></tr><tr><td> TrypLE ExpressのW / Oフェノールレッド</td><td> Gibco社、Invitrogen社</td><td> 12604 </td></tr><tr><td>シトシン-β-D-アラビノフラノシド塩酸塩</td><td>シグマアルドリッチ</td><td> C6645 </td></tr><tr><td>ポリ-D-リジン</td><td>シグマアルドリッチ</td><td> P6407 </td></tr><tr><td>ラミニン1 mg / mlの</td><td>ミリポア</td><td> CC095 </td></tr><tr><td> HEPES </td><td>シグマアルドリッチ</td><td> H3375 </td></tr><tr><td>トリパンブルーは0.4％の染色</td><td> Gibco社、Invitrogen社</td><td> 15250 </td></tr></tbody></テーブル&gt; 表IV。特定の試薬 ​​が含まれています 。 <table border="1"><tbody><tr><td> 機器 </td><td> 会社 </td><td> 猫。数 </td></tr><tr><td>ステレオ顕微鏡</td><td>オリンポス</td><td> SZ61 </td></tr><tr><td>大鉗子</td><td> FST </td><td> 11022から14 </td></tr><tr><td>先の細いピンセット</td><td>モリア</td><td> MC40B </td></tr><tr><td>マイクロ先の細いピンセット</td><td>モリア</td><td> MC31 </td></tr><tr><td>鋭いはさみ</td><td> Roboz </td><td> RS-6820 </td></tr><tr><td>ミクロ解剖はさみ</td><td> FST </td><td> 91460から11 </td></tr><tr><td> Microは、湾曲したハサミの解剖</td><td> FST </td><td> 14067から11 </td></tr><tr><td>ガラス2 - チャンバースライド</td><td>ラボ·テック</td><td> 154461 </td></tr><tr><td> 60 mmの皿</td&gt; <td> BDファルコン</td><td> 353002 </td></tr><tr><td> 100ミリメートル料理</td><td>コー​​ニング</td><td> 430167 </td></tr><tr><td> 15 mlチューブ</td><td> BDファルコン</td><td> 352099 </td></tr><tr><td> 1.5ミリリットルクライオチューブバイアル</td><td>ヌンク</td><td> 375353 </td></tr></tbody></table> 表V.特定の機器 。

isolation and culture of rat embryonic neural cells: a quick protocol

我々は、E15-17ラットの胚から海馬または皮質ニューロンを分離し、培養するための簡単​​な方法を記述しています。手順は、マウスとヒトの一次ニューロンと神経前駆細胞の分離に正常に適用することができます。解離ニューロンは、数週間に、無血清培地に維持されます。これらの培養物は、ヌクレオ、免疫細胞化学、核酸の調製と同様に、電気生理学的に使用することができます。古いニューロンの培養物は、またリン酸カルシウムや、リポフェクトアミンなどの脂質ベースの方法で、効率は低下し、レンチウイルス形質導入により効率の良いレートでトランスフェクトすることができます。

Watch this Scientific Journal Video about Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol at JoVE.com

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

我々は、齧歯類の胚から海馬および皮質ニューロンを分離、培養するための迅速な方法論を記述します。このプロトコルは、私たちはほぼ純粋な神経細胞の培養が必要とされる実験を行うことができます。

ラット胚神経細胞の分離と文化：クイックプロトコル

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

isolation-and-culture-of-rat-embryonic-neural-cells-a-quick-protocol

Research

JoVE Journal

Neuroscience

38.0K ビュー.  LSU Health Sciences Center - New Orleans. 我々は、齧歯類の胚から海馬および皮質ニューロンを分離、培養するための迅速な方法論を記述します。このプロトコルは、私たちはほぼ純粋な神経細胞の培養が必要とされる実験を行うことができます。

ビデオ: Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

胚の脊髄から交連ニューロンの解剖と文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

神経科学

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

ニューロスフェアのアッセイを使用して成体マウス神経幹細胞の単離と拡大

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

ニューロスフェアのアッセイを使用してマウス胎児神経幹細胞培養の確立

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

人間の毛包から神経堤幹細胞の単離および培養

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. Neuroepithelial cells of the neural tube that express Nkx6.1 are the unique precursor cells for spinal motoneurons1. Though postmitotic motoneurons move towards their final position and organize themselves into columns along the spinal tract2,3. More than 90% of all these differentiated and positioned motoneurons express the transcription factors Islet 1/2. They innervate the muscles of the limbs as well as those of the body and the inner organs. Among others, motoneurons typically express the high affinity receptors for brain derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT-3), the tropomyosin-related kinase B and C (TrkB, TrkC). They do not express the tropomyosin-related kinase A (TrkA)4. Beside the two high affinity receptors, motoneurons do express the low affinity neurotrophin receptor p75NTR. The p75NTR can bind all neurotrophins with similar but lower affinity to all neurotrophins than the high affinity receptors would bind the mature neurotrophins. Within the embryonic spinal cord, the p75NTR is exclusively expressed by the spinal motoneurons5. This has been used to develop motoneuron isolation techniques to purify the cells from the vast majority of surrounding cells6. Isolating motoneurons with the help of specific antibodies (panning) against the extracellular domains of p75NTR has turned out to be an expensive method as the amount of antibody used for a single experiment is high due to the size of the plate used for panning. A much more economical alternative is the use of lectin. Lectin has been shown to specifically bind to p75NTR as well7. The following method describes an alternative technique using wheat germ agglutinin for a preplating procedure instead of the p75NTR antibody. The lectin is an extremely inexpensive alternative to the p75NTR antibody and the purification grades using lectin are comparable to that of the p75NTR antibody. Motoneurons from the embryonic spinal cord can be isolated by this method, survive and grow out neurites.

An alternative way of isolating mouse embryonic motoneurons from the spinal cord is described. The method takes into account the fact that lectin can bind to the low affinity nerve growth factor receptor p75NTR. This lectin-based preplating allows a purification similar to that with a specific antibody against the p75NTR.

マウス胚の運動ニューロンのレクチンに基づく分離と文化

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. ...

Isolation and Culture of Human Fungiform Taste Papillae Cells

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices1,2,3,4. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d5,6,7,8. We recently reported on a successful method for the extended culture of rodent taste cells9. We here report for the first time the establishment of an in vitro culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C &beta;2, (PLC-&beta;2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-&beta;2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro preparation.
Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al. 201110.

We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability.

人間の茸状の味覚乳頭細胞の単離および培養

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

胎児マウス神経管から神経堤細胞の単離および培養

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

の解離、文化、および分析<em&gt;アフリカツメガエル</em&gt;胚性網膜組織

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

胚前脳からの内皮細胞の単離および培養

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

マウスを一回、二つの文化：個々のマウスの二つの神経原性のゾーンから成体神経幹細胞の単離および培養

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...