Name: organotypic hippocampal slice cultures as a model to study neuroprotection and invasiveness of tumor cells
Uploaded: 2017-08-27T13:00:00.000+00:00
Duration: 7 min 48 s
Description: Watch this Scientific Journal Video about Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells at JoVE.com

著者は、優れた技術的な援助のためのビデオ録画とハリッド Ghadban 彼女を扶養するクリスティン Auste を感謝したいです。ウルシュラ Grabiec は、ルーの Programm FKZ 29/18 によって支えられました。

によってヨーロッパ コミュニティ評議会指令 2010年/63/EU 欧州議会を承認済みとして倫理ポリシーおよび神経科学研究の動物の使用に関する方針に従って動物実験を行ったと科学的な目的に使用される動物の保護に関する欧州連合理事会の。
 1 器具文化メディア準備 <ol><li>の一部は、OHSC 準備のため楽器の次のセットを使用して、: 2 つの小さなはさみ、2 つピンセット、繊細なティップを持つ 1 つのピンセット (サイズ 11、15 のサイズの 1 つの 2 つ)、3 つのブレード。3 メス ホルダー、ラウンド フィルター ペーパー (直径: 35 mm)、寒天、1 つのかみそりの刃、パスツール ピペット、未変更の状態の 1 つであり、1 つ変更パスツール ピペット チップなし。( 図 1) 使用前にオートクレーブのすべての材料を滅菌します</li>。
	<li>寒天 5 g の重量を量るし、100 ml の蒸留水に溶かします。滅菌オートクレーブ 210.8 kPa 121.7 ° C で 20 分のためのソリューション。滅菌ガラス ピペットを使用して 60 mm ペトリ皿で 3 mL 液体寒天液を配布、5 h を固めるは、汚染を避けるため、さらに使用するまで 4 ° C で保存しプラスチック パラフィン フィルムでカバーすること。寒天ブロックがスライスのプロシージャの間に脳を安定させるために必要な</li>。
	<li>メディア <ol><li>準備中 (pH 7.35)、200 mL で 198 mL 最小必須培地 (MEM)、2 mL L グルタミン ソリューション (最終濃度 2 mM)。メディアの準備の日にソリューションを準備し、4 で保存 ° C</li>
		<li>準備 49 mL MEM、25 mL ・ ハンクスから成る培養液 100 mL &#39; バランスのとれた塩溶液 (HBSS)、25 mL (v/v) 通常の馬血清 (NHS)、L-グルタミンの解決の 1 mL (最終濃度 2 mM)、100 μ g インスリン、血糖値 120 mg 10 mg ストレプトマイシン、10,000 U ペニシリンと 800 μ g ビタミン C 既に報告されています。ミディアム (37 ° C) を暖かい、pH 7.4 で滅菌フィルター (0.2 μ m 孔サイズ) に pH 値を調整します。媒体を変更する前にすべての 2 日目 (ウォーミング アップ、pH 調整等) 手順を繰り返します。1 週間で最もメディアを使用 4 で保存する場合 ° C</li>
	</ol></li> <li>脳を格納する準備の中で 1 つの 35 mm のペトリ皿を埋めます。作業領域の冷却パックに組織を収集するため 2 空のシャーレを配置します</li>。
</ol> 2。準備と、Vibratome とスライス <ol><li>使用脳 7-9 日古いラットからまたは 4-5 日 Stoppini ら によると一部は、OHSC 準備のため古いマウスします 。4 動物の斬首後、は、はさみで、頭蓋骨から皮膚を削除する。</li>。
	<li>は後頭に導入して高級シザーの刃と尾側吻側 (奥) (フロント) 軸に沿って切断して頭蓋骨を開きます。2 つ作るは、それぞれ左と右耳に向かってハサミを指すので、最初の 1 つに垂直をカット (&#34; T 切開 &#34;).</li>
	<li>は、脳を傷つけないように注意を払って、高級ピンセットで慎重に頭蓋骨を開きます。前頭葉と小脳の最も吻側部をカットするメス (ブレード サイズ 11) を使用します</li>。
	<li>はヘラの手段で脳を削除し、準備中 ( 図 2) でいっぱいペトリ皿に慎重に配置します。試料ホルダーに脳を置き、医療シアノアクリ レート系接着剤で固定します。機械的な安定を確保するために、寒天の部分を使用します</li>。
	<li>350 μ m で水平方向に組織を解剖スライド式 vibratome を使用して厚い一部は、OHSC</li>。
	<li>は、光学双眼顕微鏡を使用してスライスを評価します。低品質の一部は、OHSC をすぐに破棄します。海馬の中央部から分離されたそのままの細胞構築とそれらのスライスのみを取ることが重要です (図 3 および図 4 を参照)、背側と腹側海馬と</li>。
	<li>別の海馬領域と (丸刃サイズ 15; メスによる嗅 図 3)。貫通経路と嗅皮質を保持する必要があります</li>。
	<li>一部は、OHSC 6 ~ 8 はそれぞれの脳から取得されます。2-3 スライスを 1 つのセルの文化挿入 (細孔サイズ 0.4 μ m) に転送し、ウェルあたり 1 mL の培養液を含む 6 ウェル培養皿の 1 つもです</li>。
	<li>5% (v/v) CO 2 と完全に加湿雰囲気で 35 ° C で 6 よく料理を孵化させなさい、細胞培養培地は 2 番目毎日変更します 。
	注: は、6 日 体外 (div) にあなたの実験を行います。6 日目でスライスのプロシージャに関連付けられている炎症反応が消えてしまいます。再びミクログリア細胞の表示区分された形態をこの段階で、シナプスが成熟している</li>。
</ol> 3。組織品質の評価 <ol><li>固定、ラベリングと可視化の一部は、OHSC 変性ニューロン <ol><li>実験を実行した後で 5 μ G/ml propidium ヨウ化 (PI)、固定する前に 2 時間、一部は、OHSC を孵化させなさい。変性神経細胞の核を染色する順序 。
		注意: PI は疑わしい発癌物質、常に手袋などの個人保護用具 (PPE) を着用します</li>。
		<li>0.1 M リン酸緩衝液 (7.4) と、一部は、OHSC を洗う、パラホルムアルデヒド (PFA) 24 時間のための 4% (v/v) 溶液で固定 
		注意: PFA は有害と疑われる発がん性物質です。ヒューム フードの下で動作し、PPE を着用します</li>。
		<li>1 ml の PBS の挿入で、一部は、OHSC を洗うし、リガー ブラシを使用して、スライスを細胞膜から分離します</li>。
		<li>染付 IB 4 24 ウェル プレート ( 図 5) で、一部は、OHSC のラベルします</li>。
	</ol></li> <li>蛍光を用いた腫瘍侵入実験の伝導標識単一細胞 <ol><li>の実験開始前に 24 h 蛍光色素 Carboxyfluorescein アセテート染色による腫瘍細胞にラベルを付けるエステル (CFDA SE).</li>
		<li>デタッチ ノイバウアー チャンバーを用いた腫瘍細胞を数えると</li>。
		<li>懸濁液の 10 μ L が目的のセル数 (通常 50,000 または 100,000 細胞) を含むような培地で細胞を再懸濁します</li>。
		<li>3 日間侵入する細胞ができ、スライス培養に細胞懸濁液の 10 μ L を適用します</li>。
		<li>実験の最後に、4% (v/v) PFA、24 時間を使用してスライス培養を修正し、細胞構築を可視化する別の 24 h の PI と共培養のラベルします 。
		注意: PFA は有害と疑われる発がん性物質です。ヒューム フードの下で動作し、PPE を着用します</li>。
		<li>メディアをマウントを使用してさらに分析のカバー スリップの上に共培養をマウントします</li>。
	</ol></li></ol> 4。一部は、OHSC 実験の評価 共焦点レーザー走査顕微鏡 (CLSM) で、一部は、OHSC を分析します。円周率の検出ラベル、ニューロンの変性または細胞構築をラベル PI 使用波長 λ の単色光 = 543 nm、発光帯の波長 λ のためのフィルターを渡す = 585 615 nm。腫瘍細胞または IB 4 ミクログリアというラベルの付いた、CFDA、励起波長 λ を使用して = 488 nm。両方の実験の種類は、2 μ m の光学的スライス厚と z スタックを記録し、評価に使用します。

著者がある何も開示するには

この議定書では、一部は、OHSC の準備について説明します。このモデルは、組み込み機能と脳の反応の生理学的および病理学的刺激の適用後テストできます。ほかに電気生理学的パラメーターの解析では、一部は、OHSC は破壊することができます、あらゆる細胞に損傷の影響を決定できます。さまざまな物質と示したプロセスやマクロファージとリンパ球の不在で癒しの詳細な説明と治療が可...

一部は、OHSC 神経細胞、アストロ サイト、ミクログリア1の生理学的および病理学的性質の研究によ特徴付けられた生体外モデルであります。細胞外の環境をコントロールし、様々 な刺激の後細胞と形態学的変化を監視する簡単です。後準備2,3海馬ニューロンの組織とその接続がよく保存されています。いくつかの利点の一部は、OHSC は、動物の外科手術することがなく脳損傷およびがんの浸潤の監視を許可します。一部は、OHSC 6 〜 8 は、単一の齧歯動物の脳から入手できます。一部は、OHSC したがって大幅動物の数を減らすし、同じ動物では複数の薬物濃度、遺伝子操作または異なる病変モデルをテストできるようにするのに役立ちます。スライス ベースのアッセイでは、実験条件を正確に制御できます。また、二次被害のような病理学の条件の時間依存の開発はタイムラプス イメージングによる簡単に監視できます。
Stoppiniらによって最初に確立された特定のプロトコルに4、準備手順が記載されている、適切なスライスの選択の重要な形態学的なランドマークが強調表示されます。生後 7-9 ラットやマウス生後 4-5 の準備をお勧めします。これらの期間は、一部は、OHSC は、機械的外傷、神経回路の再編成の可能性が高い堅牢な抵抗性を示します。対照的に、萌芽期または成人ラットから準備急速に構造変更し栽培中の脊髄の形態を失う、したがって基礎研究5,6で長期的なプロセスを学ぶには適して,7,8,9,10,11. 一部は、OHSC の生存率のもう一つの重要なポイントは、拡散およびこうして栄養供給が限られた12,13,14スライス自体の厚さ。

<table><tbody><tr><td>6-Well</td><td>Falcon</td><td>35-3046</td><td></td></tr><tr><td>Agar</td><td>Fluka</td><td>5040</td><td></td></tr><tr><td>Autoclav</td><td>Systec</td><td>DX-45</td><td></td></tr><tr><td>CFDA</td><td>&amp;nbsp;Thermo Fisher</td><td>V12883</td><td></td></tr><tr><td>Confocal laser scanning microscope (CLSM) LSM700</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Eagle&amp;acute;s Minimal Essential Medium&amp;nbsp;</td><td>Invitrogen</td><td>32360-034</td><td></td></tr><tr><td>Fluorescein labeled Griffonia (Bandeiraea) Simplicifolia Lectin I</td><td>Vector Labs</td><td>FL-1101</td><td></td></tr><tr><td>Glucose</td><td>Merk</td><td>1083371000</td><td></td></tr><tr><td>Glutamin</td><td>Invitrogen</td><td>25030-024</td><td></td></tr><tr><td>Hank&amp;acute;s Balanced Salt Solution (with Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt;)</td><td>Invitrogen</td><td>24020-133</td><td></td></tr><tr><td>Hank&amp;acute;s Balanced Salt Solution (without Ca&lt;sup&gt;2+&lt;/sup&gt;&amp;nbsp; and Mg&lt;sup&gt;2+&lt;/sup&gt;)</td><td>Invitrogen</td><td>14170-138</td><td></td></tr><tr><td>Insulin</td><td>Sigma Aldrich</td><td>I5500</td><td></td></tr><tr><td>L-ascorbic acid</td><td>Sigma Aldrich</td><td>A5960</td><td></td></tr><tr><td>L-Glutamin</td><td>Invitrogen</td><td>25030-024</td><td></td></tr><tr><td>LN229</td><td>Cell-Lines-Service</td><td>300363</td><td></td></tr><tr><td>Medical cyanoacrylate glue (Histoacryl glue)</td><td>B.Braun</td><td>1050052</td><td></td></tr><tr><td>Millicell Culture Inserts</td><td>Millipore</td><td>PICMORG50</td><td></td></tr><tr><td>NMDA N-methyl-D-aspartic acid</td><td>Sigma Aldrich</td><td>M3262</td><td></td></tr><tr><td>Normal Horse Seum</td><td>Invitrogen</td><td>26050-088</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>Petri dishes (all sizes)</td><td>Greiner</td><td>627160/664160/628160</td><td></td></tr><tr><td>PFA</td><td>Roth</td><td>0335.1</td><td>toxic</td></tr><tr><td>Propidium iodid (PI)</td><td>Sigma Aldrich</td><td>81845-25MG</td><td>toxic</td></tr><tr><td>U138</td><td>ATCC</td><td>HTB-14</td><td></td></tr><tr><td>Vibratome</td><td>Leica</td><td>Leica VT 1200</td><td></td></tr></tbody></table>

organotypic hippocampal slice cultures as a model to study neuroprotection and invasiveness of tumor cells

切片海馬スライスの文化 (一部は、OHSC) でニューロンとグリア細胞の形態学的および機能的な特性がよく保存されています。このモデルは、神経保護に関する研究、神経細胞、神経回路、または浸潤に関する電気生理学的実験を伴う研究問い合わせへの対応に適しています。海馬のアーキテクチャと海馬回路における神経細胞の活動がよく保存されている一部は、OHSC、にもかかわらずスライス手順自体初期病変とグリア瘢痕の形成につながる。など、小さな分子の拡散挙動と機械的性質、瘢痕形成はおそらく変更します。スライスは、動物の外科手術、各種脳由来細胞、アストロ サイト、ミクログリア、神経生理学的および病理学的下すなわち間相互作用の脳損傷後の時間依存プロセスの監視を許可します。条件。このモデルの相反する側面血流および血液の免疫細胞の不在であります。神経障害の進行、中に血液が重要な役割を果たすから免疫細胞の移行。それらの細胞はスライスで行方不明、文化の本質的なプロセスが外部に干渉しないで観察可能性があります。また、一部は、OHSC 媒体外部環境の組成物は、正確に制御されます。このメソッドのさらなる利点は、標準製剤と比較して犠牲にされた動物の数が少ないです。いくつか一部は、OHSC は 1 頭の動物に複数の治療法を同時研究を可能 1 つの動物から入手できます。これらの理由の一部は、OHSC は組織の損傷後や腫瘍の侵入の間に新しい保護治療の効果を分析する適しています。
提示プロトコルの一部は、OHSC 再現性の高い生成することができます準備方法を記述するここで、神経保護や腫瘍浸潤の研究のような実験的研究のさまざまな使用することができますスライスをよく保存します。

神経保護の研究:神経損傷、PI 肯定的な核と IB4 (DG) 歯状回の顆粒細胞層 (GCL) のすべての 3 番目の光学セクションにおけるミクログリアに数えられた正の数を確認します。腫瘍の侵入実験のためスタックの最大強度 z 投影は侵略と異なる侵入パターン (図 6) を視覚化する手段として腫瘍細胞によって覆われる領域を計算するた...

Watch this Scientific Journal Video about Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells at JoVE.com

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

切片海馬スライス培養 (一部は、OHSC) は、体内の状況を非常によくシミュレートする生体外モデルを表します。ここで述べる vibratome ベース新規物質の神経電位または腫瘍細胞の生物学的挙動の評価で使用するための高品質のスライスを取得するためのプロトコルをスライスを改善します。

研究保護および腫瘍細胞の侵襲性をモデルとして脊髄海馬スライス培養

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

organotypic-hippocampal-slice-cultures-as-model-to-study

Research

JoVE Journal

Neuroscience

12.9K ビュー.  Martin Luther University Halle-Wittenberg. 切片海馬スライス培養 (一部は、OHSC) は、体内の状況を非常によくシミュレートする生体外モデルを表します。ここで述べる vibratome ベース新規物質の神経電位または腫瘍細胞の生物学的挙動の評価で使用するための高品質のスライスを取得するためのプロトコルをスライスを改善します。

ビデオ: Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

生後神経発生の研究のための器官型スライス培養

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

発生生物学

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

神経損傷を調べるための器官型海馬スライス培養モデル

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

神経科学

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

器官型海馬切片培養

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7.
Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8.
For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.

The organotypic brain slice coculture with carcinoma cells enables visualizing morphological changes by fluorescence as well as bright field (video) microscopy during the process of carcinoma cell invasion of brain tissue. This model system also allows for cell exchange and replenishment approaches and offers a wide variety of manipulations and analyses.

器官型脳スライスと癌細胞の3Dスフェロイドとの共培養システム

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in ...

医学

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

Entorhino海馬器官型スライス培養における神経血管リモデリングの解析

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

腫瘍微小環境と標的薬物療法研究のための脳の腫瘍/器官スライス共培養系

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...

A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor invasion into surrounding brain parenchyma represents an enduring therapeutic challenge. To develop anti-migration therapies for GBM, model systems that provide a physiologically relevant background for controlled experimentation are essential. Here, we present a protocol for generating slice cultures from human GBM tissue obtained during surgical resection. These cultures allow for ex vivo experimentation without passaging through animal xenografts or single cell cultures. Further, we describe the use of time-lapse laser scanning confocal microscopy in conjunction with cell tracking to quantitatively study the migratory behavior of tumor cells and associated response to therapeutics. Slices are reproducibly generated within 90 min of surgical tissue acquisition. Retrovirally-mediated fluorescent cell labeling, confocal imaging, and tumor cell migration analyses are subsequently completed within two weeks of culture. We have successfully used these slice cultures to uncover genetic factors associated with increased migratory behavior in human GBM. Further, we have validated the model&#39;s ability to detect patient-specific variation in response to anti-migration therapies. Moving forward, human GBM slice cultures are an attractive platform for rapid ex vivo assessment of tumor sensitivity to therapeutic agents, in order to advance personalized neuro-oncologic therapy.

Current ex vivo models of glioblastoma (GBM) are not optimized for physiologically relevant study of human tumor invasion. Here, we present a protocol for generation and maintenance of organotypic slice cultures from fresh human GBM tissue. A description of time-lapse microscopy and quantitative cell migration analysis techniques is provided.

腫瘍細胞移動の研究および抗浸潤性薬物の患者特異的効果の研究のためのヒト神経膠芽腫の器官型スライス培養モデル

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor ...

Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early postnatal development, cerebellar Purkinje cells are known to go through a vulnerable period, during which they undergo programmed cell death. Here, we provide a detailed protocol to perform mouse organotypic cerebellar slice culture during this critical time. The slices are further labeled to assess Purkinje cell survival and the efficacy of neuroprotective treatments. This method can be extremely valuable to screen for new neuroactive molecules.

Organotypic slice cultures are a powerful tool to study neurodevelopmental or degenerative/regenerative processes. Here, we describe a protocol that models the neurodevelopmental death of Purkinje cells in mouse cerebellar slice cultures. This method may benefit research in neuroprotective drug discovery.

オルガ鼻記小脳スライス培養におけるプルキンジェ細胞生存

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early ...

A Model of Epileptogenesis in Rhinal Cortex-Hippocampus Organotypic Slice Cultures

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s neuroprotective and therapeutic potential. Organotypic slices are prepared from explanted tissue and represent a complex multicellular ex vivo environment. They preserve the three-dimensional cytoarchitecture and local environment of brain cells, maintain the neuronal connectivity and the neuron-glia reciprocal interaction. Hippocampal organotypic slices are considered suitable to explore the basic mechanisms of epileptogenesis, but clinical research and animal models of epilepsy have suggested that the rhinal cortex, composed of perirhinal and entorhinal cortices, play a relevant role in seizure generation.
Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Recordings of spontaneous activity from the CA3 area under perfusion with complete growth medium, at physiological temperature and in the absence of pharmacological manipulations, showed that these slices depict evolving epileptic-like events throughout time in culture. Increased cell death, through propidium iodide uptake assay, and gliosis, assessed with fluorescence-coupled immunohistochemistry, was also observed. The experimental approach presented highlights the value of rhinal cortex-hippocampus organotypic slice cultures as a platform to study the dynamics and progression of epileptogenesis and to screen potential therapeutic targets for this brain pathology.

Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Under a gradual and controlled deprivation of serum, these slices depict evolving epileptic-like events and can be considered an ex vivo model of epileptogenesis. This system represents an excellent tool for monitoring the dynamics of spontaneous activity, as well as for assessing the progression of neuroinflammatory features throughout the course of epileptogenesis.

リナル皮質-海馬の組織性スライス培養におけるてんかん発生のモデル

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s ...

Establishing a Brain Tumor in an Organotypic Slice Co-culture Model

Source: Chadwick, E. J. et al. <a href="https://app.jove.com/v/53304">A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies.</a> J. Vis. Exp. (2015).
This video demonstrates the development of a 3D co-culture system. Mouse brain slices are placed on a membrane, and fluorescently labeled human brain tumor cells are allowed to grow on it. Once the system is ready, it is stained for imaging.

器官型スライス共培養モデルにおける脳腫瘍の確立

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Plating the Slices onto the ...