この研究は、UCIの学部研究機会プログラムからの補助金（ARとSP）とカリフォルニアアルツハイマー病のイニシアティブと保健助成金の国民の協会がないの国からの補助金によって支えられて。 HD38466、およびアルツハイマー病研究センターは付与しない。 AG16573（JB）

1。皮質組織ブロックの凍結保存 材料の準備 <ol><li>滅菌水で10倍ストック溶液（1:9）で希釈することにより1Xハンクスバッファード塩類溶液（HBSS）を準備する。 4℃でHBSSを保管してください。 </li><li> HBSS（1:9）でDMSOを希釈して10％DMSOを凍結培地を準備します。凍結培地は、凍結保存前の新鮮なものとチルドまで4℃で保存してください。 </li><li>鋼のカミソリの刃は、組織の体系的チョッピングのために使用されます。使用前に、カミソリの刃は2時間、70％エタノールに浸して殺菌される。すぐに組織の処理の前に、滅菌水で3回カミソリの刃を洗う。それが酸化されやすいため、時間の過度の量の滅菌水にカミソリを放置しない。 </li><li> Nalgeneの冷凍コンテナはcryovials（2.5mL）でロードされ、無菌生物学的なキャビネットに入れられます。チルド凍結培地1mlを各バイアルに追加されます。冷凍コンテナは、少なくとも2時間4℃で配置されます。 </li></ol> 、クリーニングチョッピング、および凍結 <ol><li>フリーズする脳組織は、髄膜と血管の洗浄される。アイスパックの上で作業しながら慎重に組織からごみを削除するには、滅菌針のヒントを参考にしてください。洗浄組織は冷HBSSで軽くすすぎ、チョッピングのための新しいシャーレに転送する必要があります。 </li><li>アイスパックの上部に取り組んで、急速に約1mm 3ブロックに組織を分割する場合に滅菌カミソリの刃を使用してください。より均一なブロックサイズで、その結果、チョッピングの手順を適切に制御における時間の結果で洗浄組織の小さな部分での作業。 </li><li>徐々に50 mLコニカルチューブにHBSS 50mlに切り刻まれた組織を追加します。ゆっくりと、残りの組織ブロックを収集するために、HBSSでペトリ皿を洗う。緩くパックされた組織ブロックペレットを作成し、組織ブロックをチューブの底に下降することができます。 </li><li>組織がセトリングしている間、生物学的安全キャビネット内に以前にチルド冷凍コンテナとcryovialsをもたらす。組織を追加するプロセスを早めるために、すべてのcryovialsを暴露する。 </li><li>すべての組織ブロックが定着した後、軽くペレット上記のメディアの非常に薄い層を残して、余​​分なHBSSを吸引除去する。それが組織ブロックは互いに、大幅に減少冷凍効率に付着する原因となります遠心分離することは推奨されません。 </li><li>ゆっくりと広い開口部（滅菌済みのハサミで先端をカット）とピペットチップを使用して緩い組織ブロックペレットの底から200μLを収集する。 oneクライオバイアルに材料を移し、ペレットの底から次の、もう一度収集組織に移ります。それは全体の組織の割り当ての手順は、冷凍コンテナ3〜4分以上をとらないことが重要です。これは組織が凍結する前に時間の短期間だけDMSOにさらされることが保証されます。バイアルに組織を転送した後、少なくとも4時間、80℃冷凍庫で冷凍コンテナを配置します。また、凍結容器を-80℃で一晩放置することができます。残りの冷凍コンテナ用にこのプロセスを繰り返します。 </li><li>冷凍コンテナ（複数可）から長期保存用液体窒素タンクでcryoboxと場所にcryovialsを転送します。 </li></ol> 2。融解と凍結皮質組織ブロックの培養 材料の準備 <ol><li>前に解凍組織サンプルへの一日は、ポリ- L -リジンコートしたペトリ皿/カバースリップを用意しています。一般的には、皮質ニューロンのために我々は500μg/ mLのまたは1mgの/ mLでコーティングされた料理を準備。完全に皿の底をカバーするのに十分なポリ- L -リジン溶液を（ホウ酸緩衝液で作られた）を追加します。ガラス製カバースリップが必要な場合、それらは完全にポリ- L -リジン溶液で浸漬されていることを確認してください。少なくとも12時間インキュベートする。ご使用の前に、徹底的に滅菌水で3回、5分ごとのリベラルな量の料理をすすぐ。 </li><li>暖かいHBSSは、細胞懸濁液に組織を解離するだけでなく、解凍した組織をきれいにするために使用されます。必要なHBSSの量は、融解する組織の量によって異なりますが、通常は1クライオバイアルは、HBSSを50mLに希釈し、HBSSを10mLの解離になります。 </li><li> 10％の鉄添加仔ウシ血清（EBS）と1％のダルベッコ変法イーグル培地抗生物質/抗真菌剤（AA）混合物（DMEM (++))は37℃の水浴中に配置する必要があります。 </li><li>メッキの培地（NB (+++))は使用前に新鮮なされるべきである。この培地は、抗生物質/抗真菌剤に加えてB27とN2のサプリメントを1％添加することによって調製される。 </li></ol> 注：十字（+）を付加したメディア名がメディアの塩基組成に添加剤を含めることを示す。このテキストでは、DMEM（+ +）を意味するDMEM + 10％EBSプラス1％AA、一方、NB (+++)で囲まNB PL私たち一パーセントAAプラスB27プラスN2。 解凍と培養 <ol><li>液体窒素タンクからcryovialsを削除し、急速に37℃で融解°のみ小さなアイスペレットになるまでの水浴中でCがバイアルの内容の内部に観察される。 </li><li>静かにコニカルチューブに暖かいHBSS 50mlのバイアルの内容を転送する。優しくクライオバイアルにはまり、残りの組織ブロックを取り除くために、広い開口部の先端を使用してください。チューブを3-4回転倒し、組織が徐々に下部に収集することができます。組織は、迅速に解決するはずですが、ブロックが小さすぎる場合、これは推奨されている遠心分離（〜200 × g）を発生し、点灯しないことがあります。 </li><li>過剰なHBSSを吸引除去する。 </li><li>組織ペレットと、0.25％トリプシンとDNAase 50μL、300μLに暖かいHBSSを10mLを追加。 5分間37℃の水浴中でインキュベートし、その後、軌道シェーカーの場所は、° C、さらに5分間に80回転と37に設定。 </li><li>この期間の後、生物学的安全キャビネット内に組織ブロックを持参し、曇った細胞懸濁液が形成されるまで慎重に組織ブロックを撹拌するために10 mLのピペットを使用してください。暖かいDMEM 10mLを加えることによってトリプシンを非活性化（+ +）細胞懸濁液に。 </li><li> 5分間1200 × gで解離細胞を遠心分離します。上清を吸引し、廃棄、暖かいDMEM 10mLに細胞ペレットを再懸濁します（+ +）と血球計数器を用いて細胞数を定量化する。 </li><li>ポリ- L -リジンコートディッシュ上に細胞をプレート。通常は、60 mmディッシュは、1 × 10 6個の細胞でメッキされます。 </li><li>細胞は組織培養インキュベーターで約1時間のための料理/カバーガラスに付着することができます。それは、効果的な添付ファイルが観察されるまでワンプレートで10分ごとに添付ファイルの進捗を監視することをお勧めします。その後、ゆっくりと新鮮なDMEM (++).で培地を交換してください</li><li> 24時間後、DMEMを交換してください（+ +）暖かいNB (+++).と部分的な媒質の変化（50％）は新鮮なNB (+++).で5日ごとに行われている健全な文化は、通常、24時間後に分化し、プロセスの成長の兆しを示しています。これらの条件下で、そしてすべての4-5日の部分培地の変更を行う、培養物まで4-6週間の時間の長時間維持することができます。 </li><li>神経前駆細胞（NPCに）の生成では、同様のプロトコルは遠心分離後、EBSのないDMEMが使用されていることを除いて使用されます。細胞はneuropheresの形成を可能にするためにB27とEGF（20 ng / mL）を補充したDMEM/F12（1:3）でT - 25フラスコに播種されています。 NPCを区別するには、ニューロスフェアは、ラミニン（10 ug / mlと）DMEM/F12（1:3）N2を添加した培地でコーティングされたカバースリップ上に播種されています。 </li></ol> 3。代表的な結果健全な文化は、5日後に解凍した後、大幅な成長と分化を表示され、通常10日間（図1）によって、その成長に安定します。これらの培養物のImmuncocytochemical染色では多数の星状膠細胞（図2A）を明らかにし、ヒトおよびラットの両方の主ニューロン培養のための神経細胞（図2B）。このプロトコルは、また高品質の混合培養（図3B、C）の結果がどの差別化条件の下で自由に浮遊ロスフェア（図3A）、などのNPCの発電に適しています。 <img src="/files/ftp_upload/2384/2384fig1.jpg" alt="図1" /> 図1。凍結ヒト皮質組織の細胞培養。凍結ヒト皮質組織ブロックを解凍し、播種ポリリジンコートしたカバースリップ上で10日間栽培されている。日までに5セルの拡張は明らかであり、一日で10コンフルエントが達成されます。スケールバー：100μmである。 <img src="/files/ftp_upload/2384/2384fig2.jpg" alt="図2" /> 図2。培養細胞の免疫細胞化学染色。（）培養物は、グリアマーカーグリア線維性酸性タンパク質（GFAP、太い矢印）で染色した。 （B）神経細胞は神経マーカーβ-チューブリンIII（TIII、細い矢印）で検出した。 （C）ともに、ヒトおよびラットの文化は文化の中で10日後の豊かなグリア細胞と神経細胞を示す。一次抗体：マウス抗GFAP（1:1000）とウサギ抗- TIII（1:1000）。二次抗体：抗マウスアレクサ594（1:500）と抗ウサギアレクサ488（1:500）。核染色：Hoestchブルー（1:1000）。スケールバー：50μmである。 <img src="/files/ftp_upload/2384/2384fig3.jpg" alt="図3" /> 図3。ニューロスフェアの世代。（）凍結組織は、上記のように処理し、ニューロスフェアとして伝播させた。 （B）ニューロスフェアは、離れてニューロスフェアから移行する細胞で、その結果、差別化の条件下でガラス製カバースリップ上にプレーティングし、10日間増殖させた。 （C）ニューロンとアストロサイトはどちらも差別化ニューロスフィアの拡大フリンジに存在している。核の対比染色、図2のように使用される一次および二次抗体。

<ol>
	<li>Robbins, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+human+brain+tissue.">Cryopreservation of human brain tissue.</a> Exp Neurol. 107, 208-213 (1990).</li><li>Ware, C. B., Nelson, A. M., Blau, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled-rate+freezing+of+human+ES+cells.">Controlled-rate freezing of human ES cells.</a> Biotechniques. 38, 879-880 (2005).</li><li>Paynter, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+and+practical+issues+for+cryopreservation+of+nerve+cells.">Principles and practical issues for cryopreservation of nerve cells.</a> Brain Res Bull. 75, 1-14 (2008).</li><li>Thirumala, S., Gimble, J. M., Devireddy, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+stromal+vascular+fraction+of+adipose+tissue+in+a+serum-free+freezing+medium.">Cryopreservation of stromal vascular fraction of adipose tissue in a serum-free freezing medium.</a> J Tissue Eng Regen Med. 4, 224-232 (2010).</li></ol>

利害の衝突は宣言されません。

冷凍保存は、将来の使用のために銀行貴重な脳組織のサンプルをする機会を提供しています。ここでは、ニューロンの濃縮培養と凍結脳組織ブロックからの神経前駆細胞の両方を生成するために単純だが効果的なプロトコルを記述する。この経済的な手順は、より高価な速度制御の冷凍庫を利用する従来の凍結保存技術のコストを回避することができます。プロトコルは、実行可能なニューロン培養組織ブロックを凍結する迅速な、まだ効果的な手段を提供することにより、後の融解の発生することができます。全体凍結のプロセスは、わずか20分かかることがあります。一次ニューロン培養に加えて、このメソッドは組織ブロックを使用しても、自由に浮遊ロスフェアとして成長NPCを生成するために解凍することができます。この点で、私たちの凍結培地中の血清の不足は、細胞が未分化状態で維持されることが保証されます。差別化の条件下では、ニューロスフェアは、凍結組織ショーの拡大と、新鮮な組織から生成されたニューロスフェアへの非常に匹敵する分化率から生産。

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Dulbecco's Modified Eagle Medium (DMEM)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11995-073</td>
<td>High gluclose 1X</td>
</tr>
<tr>
<td>Bovine calf serum supplemented</td>
<td>&nbsp;</td>
<td>HYCLONE</td>
<td>SH30072.03</td>
<td>For culture medium </td>
</tr>
<tr>
<td>Neurobasal-A Medium (1X), liquid</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>1088-022</td>
<td>&nbsp;</td>
<tr>
<td>B27 Supplement</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17504-044</td>
<td>Supplement for Neurobasal medium</td>
</tr>
<tr>
<td>N2 Supplement</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17502-048</td>
<td>Supplement for Neurobasal medium</td>
</tr>
<tr>
<td>Trypsin (10X)</td>
<td>&nbsp;</td>
<td>Cellgro </td>
<td>25-054CI </td>
<td>Tissue dissociation</td>
</tr>
<tr>
<td>Deoxyribonuclease 1 from bovine pancreas (DNase)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D4527-30KU</td>
<td>Tissue dissociation</td>
</tr>
<tr>
<td>Hanks Balanced Salt Solution (HBSS) (10X)</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>14065</td>
<td>Cleaning tissue, washes, and freezing medium</td>
</tr>
<tr>
<td>Poly-L-Lysine- 500mg</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>P2636</td>
<td>Substrate for adhesion of neuronal cells</td>
</tr>
<tr>
<td>antibiotic-antimycotic (100X), liquid</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15240-062</td>
<td>To prevent contamination</td>
</tr>
<tr>
<td>Large orifice pipette Tips (1-200 ul)</td>
<td>&nbsp;</td>
<td>Fischer Scientific</td>
<td>02-681-141</td>
<td>To prevent shear stress to cells</td>
</tr>
<tr>
<td>Graduated pipette tips (101-1000 ul)</td>
<td>&nbsp;</td>
<td>USA Scientific</td>
<td>1111-2721</td>
<td>&nbsp;</td>
<tr>
<td>21 G1 precision guide needles</td>
<td>&nbsp;</td>
<td>Beckton Dickinson</td>
<td>305165</td>
<td>To clean tissue</td>
</tr>
<tr>
<td>10 ml pipette </td>
<td>&nbsp;</td>
<td>USA Scientific</td>
<td>1071-0810</td>
<td>Individually wrapped</td>
</tr>
<tr>
<td>50 ml tubes</td>
<td>&nbsp;</td>
<td>USA Scientific</td>
<td>926-9-04</td>
<td>&nbsp;</td>
<tr>
<td>Single edge razor blade</td>
<td>&nbsp;</td>
<td>Smith Brand</td>
<td>67-0238</td>
<td>To chop tissue</td>
</tr>
<tr>
<td>60 x 15 mm polystyrene petri dish </td>
<td>&nbsp;</td>
<td>USA Scientific</td>
<td>8609-0160</td>
<td>For general culture</td>
</tr>
<tr>
<td>100 x 15 mm polystyrene petri dish </td>
<td>&nbsp;</td>
<td>USA Scientific</td>
<td>8609-0010</td>
<td>For cleaning tissue</td>
</tr>
<tr>
<td>Cryogenic box </td>
<td>&nbsp;</td>
<td>NALGENE Labware</td>
<td>5026-1010</td>
<td>&nbsp;</td>
<tr>
<td>Freezing container</td>
<td>&nbsp;</td>
<td>NALGENE Labware</td>
<td>5100-0001</td>
<td>"Mr. Frosty"</td>
</tr>
<tr>
<td>2.0 ml cryogenic vials</td>
<td>&nbsp;</td>
<td>NALGENE Labware</td>
<td>5012-0020</td>
<td>&nbsp;</td>
<tr>
<td>DMSO</td>
<td>&nbsp;</td>
<td>Fischer Scientific</td>
<td>D128-500</td>
<td>For freezing medium</td>
</tr>
<tr>
<td>Ethanol 200 proof</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E7023</td>
<td>For sterilizing razor blade</td>
</tr>		</tbody>
		</table>
		</div>

cryopreservation of cortical tissue blocks for the generation of highly enriched neuronal cultures

本研究では、我々は、高濃縮ニューロン培養を生成するために皮質脳組織ブロックの成功凍結保存と解凍のための標準化されたプロトコルの概要を説明します。このプロトコルで使用される凍結培地は、ハンクのバッファ塩溶液（HBSS）で希釈した10％のジメチルスルホキシド（DMSO）である。皮質組織のブロックは、凍結培地を含むcryovialsに転送され、徐々に速度制御された冷凍コンテナの° C / min -1ので凍結されています。解凍後の処理と一貫して10日以内に文化と神経回路網の大幅な拡大の最初の5日間に急速な神経突起の成長を示した神経細胞に富む文化を生成した凍結組織ブロックの解離。アストロサイトマーカーグリ​​ア線維性酸性タンパク質（GFAP）および神経マー​​カーβ-チューブリンクラスIIIと免疫細胞化学的染色は、文化のニューロンとアストロサイトの高い数字を明らかにした。組織ブロックの解離後の神経前駆細胞培養の世代は急速に分化する条件下でニューロンとアストロサイトの大きな数字を生成したニューロスフェアを、拡大をもたらした。この単純な凍結保存プロトコルは、神経細胞アストロサイト、および神経前駆細胞培養の後の世代のための高い柔軟性を与える皮質脳組織ブロックの迅速、効率的、かつ安価な保存、することができます。

Watch this Scientific Journal Video about Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures at JoVE.com

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

ここで、我々は、効率的な凍結保存方法を説明し、高濃縮ニューロン培養を生成するために皮質脳組織ブロックの解凍。この単純なプロトコルでは、神経細胞のアストロサイト、および神経前駆細胞培養の後の世代のための柔軟性を提供します。

高濃縮ニューロン培養の世代のための皮質組織ブロックの凍結保存

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly ...

cryopreservation-cortical-tissue-blocks-for-generation-highly

Research

JoVE Journal

Neuroscience

13.5K ビュー.  University of California, Irvine. ここで、我々は、効率的な凍結保存方法を説明し、高濃縮ニューロン培養を生成するために皮質脳組織ブロックの解凍。この単純なプロトコルでは、神経細胞のアストロサイト、および神経前駆細胞培養の後の世代のための柔軟性を提供します。

ビデオ: Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

廃棄されたヒト胎児脳組織から神経幹細胞の生成

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

神経科学

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

器官培養における神経雪崩のマルチ電極アレイの録音

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications for understanding the pathogenesis of demyelinating diseases such as Multiple Sclerosis (MS). Cellular development is commonly studied with primary cell culture models. Primary cell culture facilitates the evaluation of a given cell type by providing a controlled environment, free of the extraneous variables that are present in vivo. While OL cultures derived from rats have provided a vast amount of insight into OL biology, similar efforts at establishing OL cultures from mice has been met with major obstacles. Developing methods to culture murine primary OLs is imperative in order to take advantage of the available transgenic mouse lines. 
Multiple methods for extraction of OPCs from rodent tissue have been described, ranging from neurosphere derivation, differential adhesion purification and immunopurification 1-3. While many methods offer success, most require extensive culture times and/or costly equipment/reagents. To circumvent this, purifying OPCs from murine tissue with an adaptation of the method originally described by McCarthy &amp; 
de Vellis 2 is preferred. This method involves physically separating OPCs from a mixed glial culture derived from neonatal rodent cortices. The result is a purified OPC population that can be differentiated into an OL-enriched culture. This approach is appealing due to its relatively short culture time and the unnecessary requirement for growth factors or immunopanning antibodies. 
While exploring the mechanisms of OL development in a purified culture is informative, it does not provide the most physiologically relevant environment for assessing myelin sheath formation. Co-culturing OLs with neurons would lend insight into the molecular underpinnings regulating OL-mediated myelination of axons. For many OL/neuron co-culture studies, dorsal root ganglion neurons (DRGNs) have proven to be the neuron type of choice. They are ideal for co-culture with OLs due to their ease of extraction, minimal amount of contaminating cells, and formation of dense neurite beds. While studies using rat/mouse myelinating xenocultures have been published 4-6, a method for the derivation of such OL/DRGN myelinating co-cultures from post-natal murine tissue has not been described. Here we present detailed methods on how to effectively produce such cultures, along with examples of expected results. These methods are useful for addressing questions relevant to OL development/myelinating function, and are useful tools in the field of neuroscience.

This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).

出生後のマウス組織から培養オリゴデンドロサイトと共培養Myelinatingニューロン/オリゴデンドロサイトエンリッチドの導出

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications ...

A Fully Automated and Highly Versatile System for Testing Multi-cognitive Functions and Recording Neuronal Activities in Rodents

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be investigated in a single task sequence. The unique feature of this system is a custom-made, acoustically transparent chamber that eliminates many of the issues associated with auditory cue control in most commercially available chambers. The ease with which operant devices can be added or replaced makes this system quite versatile, allowing for the implementation of a variety of auditory, visual, and olfactory behavioral tasks. Automation of the system allows fine temporal (10 ms) control and precise time-stamping of each event in a predesigned behavioral sequence. When combined with a multi-channel electrophysiology recording system, multiple cognitive brain functions, such as motivation, attention, decision-making, patience, and rewards, can be examined sequentially or independently.

In this report, we present a fully automated and highly versatile system capable of simultaneously testing multi-cognitive behaviors and recording neuronal activities for rodents.

マルチ認知機能のテストとげっ歯類の神経活動を記録するための完全に自動化された汎用性の高いシステム

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be ...

Preparation of Neuronal Co-cultures with Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (&#60;10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (&#62;85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.

Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.

単セルの精度で神経共培養の調製

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for &#62;24 h.

電気生理学およびカルシウムイメージングのための急性神経組織の長期のインキュベーション

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

マイクロ電極アレイにおけるニューロン細胞培養の刺激に対するネットワーク応答の時間依存的増加

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

GM-Free Generation of Blood-Derived Neuronal Cells

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other therapeutic strategies, there is currently no cure available for the injured or diseased brain. Cell replacement appears as a promising therapeutic strategy for neurodegenerative conditions. To this day, neural stem cells (NSCs) have been successfully generated from fetal tissues, human embryonic cells (ES) or induced pluripotent stem cells (iPSC). A process of dedifferentiation was initiated by activation of the novel human GPI-linked glycoprotein, which leads to generation of pluripotent stem cells. These blood-derived pluripotent stem cells (BD-PSCs) differentiate in vitro into cells with a neural phenotype as shown by brightfield and immunofluorescence microscopy. Ultrastructural analysis of these cells by means of electron microscopy confirms their primitive structure as well as neuronal-like morphology and subcellular characteristics.

We present a genetically modified-free (GM-free) method to obtain cells with a neuronal phenotype from reprogrammed peripheral blood cells. Activation of a signaling pathway linked to novel human GPI-linked protein reveals an efficient GM-free method for obtaining human pluripotent stem cells.

血液由来神経細胞のGMフリー生成

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other ...

Cone-Enriched Cultures from the Retina of Chicken Embryos to Study Rod to Cone Cellular Interactions

Human daytime vision relies on the function of cone photoreceptors at the center of the retina, the fovea. Patients suffering from the most prevalent form of inherited retinal degeneration, retinitis pigmentosa, lose night vision because of mutation driven loss of rod photoreceptors, a phenomenon followed by a progressive loss of function and death of cones leading to blindness. Geneticists have identified many genes with mutations causing this disease, but the first mutations identified questioned the mechanisms of secondary cone degeneration and how a dominant mutation in the rhodopsin gene encoding for the visual pigment expressed exclusively in rods can trigger cone degeneration.
This result of transplantations in a genetic model of the disease led to the concept of cell interactions between rods and cones and of non-cell autonomous degeneration of cones in all genetic forms of retinitis pigmentosa.
Cones comprise 5% of all photoreceptors in humans and only 3% in the mouse, so their study is difficult in these species, but cones outnumber rods in bird species. We have adapted 96-well plates to culture retinal precursors from the retina of chicken embryos at stage 29 of their development. In these primary cultures, cones represent 80% of the cells after in vitro differentiation. The cells degenerate over a period of one week in the absence of serum. Here, we describe the methods and its standardization.
This cone-enriched culture system was used to identify the epithelium-derived cone viability factor (EdCVF) by high content screening of a rat retinal pigmented epithelium normalized cDNA library. Recombinant EdCVF prevents the degeneration of the cones.

We describe a method to obtain primary cultures of cone photoreceptors from the retina of chicken embryos and its use for high content screening.

鶏胚のレチナから、ロッドからコーン細胞相互作用を研究するコーンエンリッチ培養

Human daytime vision relies on the function of cone photoreceptors at the center of the retina, the fovea. Patients suffering from the most prevalent form ...

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and large-scale genomic and drug screening. Due to the self-organizing nature of cells in brain organoids and the growing range of available protocols for their generation, issues with heterogeneity and variability between organoids have been identified. In this protocol paper, we describe a robust and replicable protocol that largely overcomes these issues and generates cortical organoids from neuroectodermal progenitors within 1 month, and that can be maintained for more than 1 year. This highly reproducible protocol can be easily carried out in a standard tissue culture room and results in organoids with a rich diversity of cell types typically found in the developing human cortex. Despite their early developmental make-up, neurons and other human brain cell types will start to exhibit the typical signs of senescence in neuronal cells after prolonged in vitro culture, making them a valuable and useful platform for studying aging-related neuronal processes. This protocol also outlines a method for detecting such senescent cells in cortical brain organoids using senescence-associated beta-galactosidase staining.

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro

In this study, we provide a detailed technique for a simple yet robust cortical organoid culture system using standard feeder-free hPSC cultures. This is a rapid, efficient, and reproducible protocol for generating organoids that model aspects of brain senescence in vitro.

インビトロでの脳ニューロン老化のモデリングのための皮質脳オルガノイドの堅牢で再現性の高い生成

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and ...