この作品は、健康助成R01DK080971と若年性糖尿病研究財団助成37-2009-44の国立研究所によってサポートされていました。また、UCSD小児糖尿病研究センター（PDRC）から受け取った支援に感謝。

1。 HEK293レイヤーのトランスフェクション<ol><li> DMEM 500mlのボトル（4.5グラム/ mlのグルコースとフェノールレッドとし、グルタミンなし）に追加することによりHEK293細胞培地を準備：50ミリリットルFBS、5ミリリットル100Xペニシリン/ストレプトマイシン溶液、5ml 100倍のL-グルタミン溶液と500μlのアムホテリシンをB. </li><li>ウェル当たりHEK293 0.5mlのメディアを用いて24ウェルプレートでHEK293細胞外板。細胞がプレートの底に均等に分散されていることを確認。 </li><li>ときHEK293細胞は、プラスミドの0.8μgのは、目的のタンパク質（哺乳動物発現ベクターに挿入）とリポフェクタミンプロトコールに従ってリポフェクタミン2000年2μlのコーディングとトランスフェ密集度100％に達する。我々は、哺乳動物細胞での発現のために設計されたpcDNA 3.3バックボーンを使用することを選択した。 </li><li>オプションとして、インキュベーションの6時間後、正常HEK293メディアとリポフェクタミンプロトコールに記載のトランスフェクションのメディアを交換します。 </li><li>トランスフェproteの発現を評価するために、に、トランスフェクトされた組織培養ウェルのサブセットは、発現タンパク質の免疫検出のために、または標準的な技術を用いてタンパク質発現のウェスタンブロット分析のために使用することができる。 </li></ol> 2。 HEK293細胞の任意の固定はトランスフェタンパク質を発現トランスフェクトしたHEK293細胞を穏やかに（また、ディスカッションを参照）HEK293細胞（ 例えば下記ステップ3.9）生きているか、またはすべてを一度にいくつかの実験のためのプレートの前に効率的な準備を可能にするために有害かもしれないメディアで共培養を容易にするために固定することができる。 <ol><li> HEK293細胞におけるタンパク質の発現の経時変化を決定するために、パイロット研究を行う。 </li><li> HEK293細胞層をトランスフェクションした後、発現の最高レベルに関連付けられているトランスフェクション後の時点で細胞から培地を吸引除去。これが最初の24時間以内に発生した場合、トランスフェクション後24時間までは吸引しないでください。 </li><li> HEを洗うD-PBSで軽くK293細胞は、次いで、500μlの4％パラホルムアルデヒド（PFA）を加え、室温で30分間インキュベートする。 （4％PFAのソリューションを作成するには、1×PBSで16％溶液を用いて開始し、1X PBSで1:4希釈してください。） </li><li>滅菌PBSを使用して、静かに4℃でPBS O / Nで細胞を3回とインキュベートを洗う</li><li>細胞を滅菌PBSで3回洗浄し、PBSを追加し、4のまま°Cを必要になるまで（最大記憶時間が発現されたタンパク質によって異なる場合があります私たちは、観察された効果を大幅に変更することなく、2週間までのニューロリギンアイソフォームを発現するようにトランスフェクトしたHEK293細胞を保存するインスリン分泌に）。 </li></ol> 3。 HEK293細胞を用いたINS-1ベータ細胞の共培養<ol><li> 50ミリリットルFBS、5ミリリットル100Xペニシリン/ストレプトマイシン、5ミリリットル100X L-グルタミン溶液、5mlのピルビン酸ナトリウム、500μlのアンホテリシン：500ミリリットルRPMI 1640（グルコースおよびフェノール赤とグルタミンなし）に追加することにより、INS-1メディアの準備B、​​500μlのβ-メルカプトエタノール（これはメディとほぼ同じですええと、通常のβ-メルカプトエタノールの添加）を除いて主要な膵島培養に用いる。 </li><li>収穫T75培養フラスコの底から約70〜80％コンフルエントでINS-1細胞、細胞ストリッパー（または別の非酵素的細胞の除去）を使用。各フラスコには、48の井戸のためにおよそ十分な細胞を提供する。 </li><li> INS-1およびHEKメディアの50/50ミックスで再懸1,200 rpmで3分間、遠心で細胞をスピンダウンした後。 INS-1細胞の70〜80％コンフルエントなフラスコは、ミクストメディア約25mlに懸濁します。 </li><li>むしろ生きたHEK293細胞よりもホルムアルデヒドで固定されたHEK293細胞を用いた場合、100％ではなく、混合メディアよりINS-1のメディアでINS-1細胞を再懸濁します。 </li><li>を10mlのピペットを用いて、均質な細胞懸濁液を作るためにペレットから細胞を取り除く。 </li><li> HEK293細胞にメディアを取り出すように吸引。 </li><li> P1000のピペットを用いて、穏やかoが側面に沿ってHEK細胞層上にINS-1細胞懸濁液500μLを加えるfをよく。毎年6井戸の後、均質細胞懸濁液を確保するために、再びINS-1細胞を再懸濁し、10ミリリットルピペットを使用しています。共培養のセットアップの全体的なスキームを図1に示されている。 </li><li>実験プロトコルに応じて24〜48時間、細胞をインキュベート。 </li><li>以上48時間共培養期間が必要な場合は、HEK293細胞を固定するために、上記のセクション2のオプションのステップを使用しています。 </li><li>プライマリー解離げっ歯類やヒト膵島 ​​を使用している場合は、5ミリリットルペン/連鎖球菌、5ミリリットルL-供給過剰となRPMIとしてその適切なメディアを確実に0.5ミリリットルアンホテリシン-Bの代わりに使用されている[ 例えば、Joveの11-15で前のプロトコルを参照してください。] INS-1メディア。 1が原因で潜在的に必要とされる島の多数の実施においては、24ウェルプレートに解離膵島細胞を使用することができますが、それは配慮​​が48ウェルプレート（ウェルあたり少ない膵島細胞を必要とする）にスケーリングまで与えられることをお勧めします。 48ウェルプレートに、解離の価値は約100の小島を追加開始するためのプレート当たりテッド細胞。解離の効果、使用方法に応じて、より少ない膵島が必要とされてもよい。 </li></ol> 4。グルコース刺激インスリン分泌<ol><li> 1時間の最低KRBで2.5 mMグルコース（クレブス - リンゲル重炭酸塩緩衝液）250μlの中の細胞をPreinc​​ubate。 、preinc​​ubate共存培地を除去し、直ちにKRBと交換する。この交換は、環境酸素にINS-1細胞の曝露を最小限にするために一度に1つのウェルを行う必要があります。お互いに低い（2.5 mM）をグルコースKRBピペッティングしながら片手で吸引。 </li><li> 2.5mMのか一度よくKRBバッファー1で20 mMグルコースどちら250μlのに為替プレインキュベーションKRB。 </li><li>具体的な実験のために適切であれば、例えば100μMのIBMXなどの追加分泌は、より堅牢な刺激性インスリン分泌応答を生成するために添加してもよい。具体的には解離した一次膵島β細胞が増加したグルコース濃度のALONに乏しい応答するeはので、ここでIBMXまたは他の分泌促進物質の添加は、特に16,17考慮すべきである。 </li><li>インキュベーションの1時間後、メディアはチューブを微量、5分間1500×gでのスピンに移す。 </li><li>上清200μlのを削除し、インスリンRIAやELISAによる分析のためにこれを使用しています。 </li><li>細胞溶解物を調製するには、プロテアーゼ阻害剤とのRIPAの細胞溶解緩衝液（150mMのNaCl、1.0％IGEPALCA-630またはノニデットP-40、0.5％デオキシコール酸ナトリウム、0.1％SDS、50mMトリス、pH8.0）を100μlのを追加すぐに4でメディアとインキュベートを除去した後プレート℃で20分間。 </li><li> 10,000 xgで15分間ライセートをスピンダウンし、インスリンRIAやELISAによる分析のために上清50μlのを削除します。 </li></ol>

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我々は、開示することは何もありません。

共培養方法は、β-細胞表面の特異的膜貫通タンパク質の、特にその細胞外ドメインの生理学的重要性を決定するために効果的な方法を提供ここで説明する。細胞表面上の関心のあるタンパク質を表示するHEK293細胞と接触で培養β細胞又はインスリノーマ細胞（例えば、ここで用いINS-1細胞など）により、実験を直接妨げることなく、細胞外タンパク質 - タンパク質相互作用の効果を決定するよ...

ここでは、特定の膜貫通タンパク質の細胞外ドメインは、インスリン分泌をどのように影響するかの調査を容易にする方法が記載されている。膵臓β-細胞表面上のタンパク質（あるいは他の分子）と目的のタンパク質との相互作用の影響方法プローブ。この方法は、ベータ細胞または他の隣接セル（ 例えば、内皮細胞、神経細胞、膵臓α細胞）によって発現細胞表面タンパク質は、細胞間の相互作用（ すなわち、隣接する表面上の相互作用パートナーとの相互作用を介し介しβ細胞機能にどのように影響するかの調査を可能にするβ細胞）。 細胞の形質膜は、細胞外環境へのブリッジとしての構造的および機能的タンパク質の複雑な配列が含まれています。経接続の形成によりまたはプラスチックシグナル伝達事象を開始することにより、細胞表面タンパク質間の相互作用は、コーディネートすることができ隣接セルの機能を有する。膵臓β細胞を膵島内にクラスタ化し、グルコース恒常性を維持するために、1協調して作用している。細胞外EPHA-ephrinAおよびグルコース刺激性インスリン分泌の調節におけるニューロリギン-2相互作用の重要性によって、例えば、明らかにし、それがますます明らかになってきている隣接表面にタンパク質との間に発生する相互作用の増大した細胞外知識ベータ細胞がインスリン分泌、β細胞機能の成熟と1-3グルコース恒常性の維持の完全な理解を得るための非常に重要になります。ここに記載された方法の目的は、特定の膜貫通または他の細胞表面結合タンパク質を含む経細胞相互作用のβ細胞機能への影響の調査を可能にすることである。異なる発現構築物でトランスフェクトしたHEK293細胞との共培養β細胞、Bの効果によって異なる細胞表面タンパク質または変異した変異体のイータ細胞機能としては、効率的に探査することができる。これは、β細胞自体をトランスフェクトすることなく達成される。 培養β細胞またはin vivoでノックダウン、ノックアウトまたは過剰発現の研究により、特定の経細胞相互作用の役割の解明は、潜在的の分析を混乱させることができる方法でβ細胞の健康及び/又は機能に影響を与え、β-細胞のmRNAおよびタンパク質の発現の直接的な摂動を必要とする特定の細胞外相互作用の効果。これらはまた、標的蛋白質の細胞内ドメインのレベルを変更し、さらに、経細胞間相互作用の影響を区別する上で、同じ細胞内のタンパク質間の相互作用による影響を許可しない近づく。ここでは、β細胞のインスリン分泌能力と応答上の特定の経細胞相互作用の効果を決定するための方法は、descrをあるアイベッド。この方法は、例えば、INS-1細胞4とインスリン分泌β-細胞株、および解離プライマリげっ歯類またはヒトβ細胞にも適用可能である。なお、HEK293（またはCOS）細胞層に発現して、特定の神経細胞のタンパク質に培養ニューロンの露出がドライブシナプス形成5,6というタンパク質を同定することができるが見つかりました神経生物学者、によって開発された共培養モデルに基づいています。神経シナプスのとβ細胞の分泌機構との間に類似点を考えると、我々は、β細胞機能を推論し、機能的成熟は、同様の経細胞の相互作用7-9によって駆動されることがあります。これらの相互作用を調査するために、我々は、β細胞が関心10のタンパク質を発現するHEK293細胞の層上に共培養された本明細書に記載のシステムを開発した。このシステムは、細胞外タンパク質 - タンパク質相互作用を操作しながらベータ細胞質をそのまま維持することができる。

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		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>pcDNA 3.3 vector/backbone</td>
			<td>Invitrogen</td>
			<td>K830001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>18324012</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Mediatech</td>
			<td>45001-312</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pen/strep solution</td>
			<td>Mediatech</td>
			<td>45001-652-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Mediatech</td>
			<td>45001-808-1/30</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Mediatech</td>
			<td>45001-404</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>D-PBS</td>
			<td>Mediatech</td>
			<td>45001-434</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Mediatech</td>
			<td>45001-710-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Invitrogen</td>
			<td>21985023</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell stripper</td>
			<td>Mediatech</td>
			<td>45000-668</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>T75 Flask</td>
			<td>BD</td>
			<td>1368065</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>50980487</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10X PBS</td>
			<td>Mediatech</td>
			<td>45001-130</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Mediatech</td>
			<td>MT35010CV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>IBMX</td>
			<td>Sigma</td>
			<td>I5879-100MG</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RIPA lysis buffer</td>
			<td>Sigma</td>
			<td>R0278</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

coculture analysis of extracellular protein interactions affecting insulin secretion by pancreatic beta cells

細胞表面タンパク質間の相互作用は隣接セルの機能を調整するのに役立ちます。膵臓β細胞を膵島内にクラスタ化し、グルコース恒常性を維持するために調整された様式で作用している。これは、隣接したβ細胞表面の膜貫通タンパク質間の相互作用は、β細胞機能の重要な決定要因であることがますます明らかになりつつある。 培養β細胞またはin vivoでノックダウン、ノックアウトまたは過剰発現の研究により、特定の経細胞相互作用の役割の解明は、潜在的影響の分析を混乱させることができる方法でβ-細胞の健康及び/又は機能に影響を与え、mRNAおよびタンパク質の発現の直接的な摂動を必要とする具体的な相互作用の。これらはまた、標的蛋白質の細胞内ドメインのレベルを変更し、DISであることが同じ細胞膜内のタンパク質間の相互作用による影響を防ぐことが近づく経細胞の相互作用の影響からtinguished。 ここでβ細胞のインスリン分泌能力と応答上の特定の経細胞相互作用の効果を決定するための方法が提示される。この方法は、例えば、INS-1細胞などのベータ細胞株、および解離プライマリβ細胞にも適用可能である。それはシナプス形成を駆動するための重要なタンパク質を同定したHEK293（またはCOS）細胞層に発現して、特定の神経細胞のタンパク質への培養神経細胞のその露出を発見した神経生物学者が開発した共培養モデルに基づいています。神経シナプスのとβ細胞の分泌機構との間に類似点を考えると、我々はβ細胞機能の成熟は、同様の経細胞の相互作用によって駆動されるかもしれないと考えた。我々は、β細胞は、目的のタンパク質を発現するHEK293細胞層上で培養されたシステムを開発した。このモデルでは、β-細胞の細胞質は、細胞外タンパク質interactioながらそのままですNSは、操作されます。我々は、主にグルコース刺激性インスリン分泌の研究に焦点を当てているが、ここで、他のプロセスを解析することができ、例えば、遺伝子発現の変化は、免疫ブロット法または定量PCRによって決定される。

メソッドを使用してここで説明する、私たちは、インスリン分泌にプロテインニューロリギンの異なる亜種の効果をテストしました。これは、β細胞機能10上ニューロリギン-2の効果を調査我々の公開されて仕事を補完する。 図2は、例えば、ここで呼ばれるニューロリギンアイソフォームを発現するようトランスフェクトHEK293細胞と共培養するINS-1ベータ細胞から得られ?...

Watch this Scientific Journal Video about Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells at JoVE.com

Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells

経細胞タンパク質相互作用は、膵臓β-細胞機能の重要な決定因子である。法適応特定貫通タンパク質がインスリン分泌にどのように影響するかを調査するためのシナプス-の共培養モデルからは、ここで詳しく説明します。トランスフェクトしたHEK293細胞は、目的のタンパク質を発現し、β細胞は、トランスフェクションまたは他の方法で直接摂動する必要はありません。

膵β細胞によるインスリン分泌に影響を及ぼす細胞外タンパク質相互作用の共培養分析

Interactions between cell-surface proteins help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within ...

coculture-analysis-extracellular-protein-interactions-affecting

Research

JoVE Journal

Medicine

12.8K ビュー.  University of California, San Diego. 経細胞タンパク質相互作用は、膵臓β-細胞機能の重要な決定因子である。法適応特定貫通タンパク質がインスリン分泌にどのように影響するかを調査するためのシナプス-の共培養モデルからは、ここで詳しく説明します。トランスフェクトしたHEK293細胞は、目的のタンパク質を発現し、β細胞は、トランスフェクションまたは他の方法で直接摂動する必?...

ビデオ: Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells

Improving IV Insulin Administration in a Community Hospital

Diabetes mellitus is a major independent risk factor for increased morbidity and mortality in the hospitalized patient, and elevated blood glucose concentrations, even in non-diabetic patients, predicts poor outcomes.1-4 The 2008 consensus statement by the American Association of Clinical Endocrinologists (AACE) and the American Diabetes Association (ADA) states that "hyperglycemia in hospitalized patients, irrespective of its cause, is unequivocally associated with adverse outcomes."5 It is important to recognize that hyperglycemia occurs in patients with known or undiagnosed diabetes as well as during acute illness in those with previously normal glucose tolerance.
The Normoglycemia in Intensive Care Evaluation-Survival Using Glucose Algorithm Regulation (NICE-SUGAR) study involved over six thousand adult intensive care unit (ICU) patients who were randomized to intensive glucose control or conventional glucose control.6 Surprisingly, this trial found that intensive glucose control increased the risk of mortality by 14% (odds ratio, 1.14; p=0.02). In addition, there was an increased prevalence of severe hypoglycemia in the intensive control group compared with the conventional control group (6.8% vs. 0.5%, respectively; p&lt;0.001). From this pivotal trial and two others,7,8 Wyoming Medical Center (WMC) realized the importance of controlling hyperglycemia in the hospitalized patient while avoiding the negative impact of resultant hypoglycemia. 
Despite multiple revisions of an IV insulin paper protocol, analysis of data from usage of the paper protocol at WMC shows that in terms of achieving normoglycemia while minimizing hypoglycemia, results were suboptimal. Therefore, through a systematical implementation plan, monitoring of patient blood glucose levels was switched from using a paper IV insulin protocol to a computerized glucose management system. By comparing blood glucose levels using the paper protocol to that of the computerized system, it was determined, that overall, the computerized glucose management system resulted in more rapid and tighter glucose control than the traditional paper protocol. Specifically, a substantial increase in the time spent within the target blood glucose concentration range, as well as a decrease in the prevalence of severe hypoglycemia (BG &lt; 40 mg/dL), clinical hypoglycemia (BG &lt; 70 mg/dL), and hyperglycemia (BG &gt; 180 mg/dL), was witnessed in the first five months after implementation of the computerized glucose management system. The computerized system achieved target concentrations in greater than 75% of all readings while minimizing the risk of hypoglycemia. The prevalence of hypoglycemia (BG &lt; 70 mg/dL) with the use of the computer glucose management system was well under 1%.

When compared to the previous paper protocol, implementation of a computerized glucose management system results in a substantial increase in blood glucose concentration measurements within the target range. Using a computerized glucose management system to monitor blood glucose levels, decreases in severe hypoglycemia (&lt;40 mg/dL), clinical hypoglycemia (&lt;70 mg/dL), and hyperglycemia (&gt;180 mg/dL) also can be observed.

コミュニティ病院でIVのインスリン投与を向上させる

Diabetes mellitus is a major independent risk factor for increased morbidity and mortality in the hospitalized patient, and elevated blood glucose ...

医学

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

単離、特徴付けと二つの異なる方法を使用して永久歯に由来するヒト歯髄幹細胞の分化の比較

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

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 ...

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from cirrhosis to cardiovascular diseases). There is no effective treatment and the in vitro tools available do not mimic the in vivo situation rendering the progress of the out of control wound healing process still enigmatic.
To date, 2D and 3D cultures of fibroblasts derived from DD patients are the main experimental models available. Primary cell cultures have many limitations; the fibroblasts derived from DD are altered by the culture conditions, lack cellular context and interactions, which are crucial for the development of fibrosis and weakly represent the derived tissue. Real-time PCR analysis of fibroblasts derived from control and DD samples show that little difference is detectable. 3D cultures of fibroblasts include addition of extracellular matrix that alters the native conditions of these cells. 
As a way to characterize the fibrotic, proliferative properties of these resection specimens we have developed a 3D culture system, using intact human resections of the nodule part of the cord. The system is based on transwell plates with an attached nitrocellulose membrane that allows contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue. No collagen gel or other extracellular matrix protein substrate is required. The tissue resection specimens maintain their viability and proliferative properties for 7 days. This is the first &#8220;organ&#8221; culture system that allows human resection specimens from DD patients to be grown ex vivo and functionally tested, recapitulating the in vivo situation.

Human Dupuytren&#039;s Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Dupuytren&#8217;s disease (DD) is a fibroproliferative disease of the palm of the hand. Here, we present a protocol to culture resection specimens from DD in a three-dimensional (3D) culture system. Such short-term culture system allows preservation of the 3D structure and molecular properties of the fibrotic tissue.

人間デュピュイトラン<em&gt;エクスビボ</em筋線維芽細胞と細胞外マトリックス相互作用の研究のために&gt;文化

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

新しい治療戦略を開発するための膵臓癌幹細胞特性を研究

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Neurodegeneration in an Animal Model of Chronic Amyloid-beta Oligomer Infusion Is Counteracted by Antibody Treatment Infused with Osmotic Pumps

Decline in hippocampal-dependent explicit memory (memory for facts and events) is one of the earliest clinical symptom of Alzheimer&#39;s disease (AD). It is well established that synapse loss and ensuing neurodegeneration are the best predictors for memory impairments in AD. Latest studies have emphasized the neurotoxic role of soluble amyloid-beta oligomers (A&#946;o) that begin to accumulate in the human brain approximately 10 to 15 yr before the clinical symptoms become apparent. Many reports indicate that soluble A&#946;o correlate with memory deficits in AD models and humans. The A&#946;o-induced neurodegeneration observed in neuronal and brain slice cultures has been more challenging to reproduce in many animal models. The model of repeated A&#946;o infusions shown here overcome this issue and allow addressing two key domains for developing new disease modifying therapies: identify biological markers to diagnose early AD, and determine the molecular mechanisms underpinning A&#946;o-induced memory deficits at the onset of AD. Since soluble A&#946;o aggregate relatively fast into insoluble A&#946; fibrils that correlate poorly with the clinical state of patients, soluble A&#946;o are prepared freshly and injected once per day during six days to produce marked cell death in the hippocampus. We used cannula specially design for simultaneous infusions of A&#946;o and continuous infusion of A&#946;o antibody (6E10) in the hippocampus using osmotic pumps. This innovative in vivo method can now be used in preclinical studies to validate the efficiency of new AD therapies that might prevent the deposition and neurotoxicity of A&#946;o in pre-dementia patients.

To study the effects of A&#946;o in vivo, we developed a model based on repeated hippocampal infusions of soluble A&#946;o coupled with continuous infusion of A&#946;o antibody (6E10) in the hippocampus using osmotic pumps to counteract the neurotoxic effect of A&#946;o.

慢性アミロイドβオリゴマー輸液の動物モデルにおける神経変性は、浸透圧ポンプを用いて抗体治療フューズによって打ち消されます

Decline in hippocampal-dependent explicit memory (memory for facts and events) is one of the earliest clinical symptom of Alzheimer&#39;s disease (AD). It ...

Application of End-to-end Anastomosis in Robotic Central Pancreatectomy

Central pancreatectomy is carried out for the treatment of benign or low-malignant potential tumors located in the pancreatic neck or proximal part of pancreatic body. With technological development, the robotic surgical system has shown its advantage in minimally invasive surgery and been increasingly applied in central pancreatectomy. However, reconstruction of the continuity of pancreas with end-to-end anastomosis after robotic central pancreatectomy has not been applied. In this study, we report surgical techniques for robotic central pancreatectomy with end-to-end anastomosis. The pancreas is reconstructed by duct-to-duct anastomosis of the pancreatic duct with a pancreatic stent inserted in the two stumps of pancreatic duct, and by end-to-end anastomosis of the pancreatic parenchyma. Compared with traditional central pancreatectomy with pancreaticoenteric anastomosis, this approach decreases the operative injury to the patient, and also conserves the integrity and continuity of the digestive duct and pancreatic duct. The robotic surgical system integrated with multiple instruments with flexible and precise movement is particularly suitable for the dissection and reconstruction of the pancreatic duct. We found that robotic central pancreatectomy with end-to-end anastomosis is safe and feasible, and we need more experience to evaluate its best indications and long-term outcomes.

The robotic central pancreatectomy with end-to-end anastomosis is feasible and safe for tumor in the pancreatic neck and proximal portion of pancreatic body. The operative techniques of this operation are presented.

ロボット中央膵の吻合をエンド ツー エンドのアプリケーション

Central pancreatectomy is carried out for the treatment of benign or low-malignant potential tumors located in the pancreatic neck or proximal part of ...

Homogeneous Time-resolved F&#246;rster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous insulin assays have existed for decades, the recent advent of homogeneous time-resolved F&#246;rster Resonance Energy Transfer (HTRF) technology has significantly simplified these measurements. This is a rapid, cost-effective, reproducible, and robust optical assay reliant upon antibodies conjugated to bright fluorophores with long lasting emission which facilitates time-resolved F&#246;rster Resonance Energy Transfer. Moreover, HTRF insulin detection is amenable for the development of high-throughput screening assays. Here we use HTRF to detect insulin secretion in INS-1E cells, a rat insulinoma-derived cell line. This allows us to estimate basal levels of insulin and their changes in response to glucose stimulation. In addition, we use this insulin detection system to confirm the role of dopamine as a negative regulator of glucose-stimulated insulin secretion (GSIS). In a similar manner, other dopamine D2-like receptor agonists, quinpirole, and bromocriptine, reduce GSIS in a concentration-dependent manner. Our results highlight the utility of the HTRF insulin assay format in determining the role of numerous drugs in GSIS and their pharmacological profiles.

Homogeneous Time-resolved F&amp;#246;rster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

Here, we present homogeneous time resolved FRET (HTRF) as an efficient method for rapid detection of insulin secreted from cells.

均質な時間分解蛍光共鳴エネルギー転送を用いた測定インスリン分泌の検出

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous ...

Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for systemic energy homeostasis. Primary hepatocytes isolated from the murine liver constitute a valuable biological tool to understand the functional properties or alterations occurring in the liver. Herein we describe a method for the isolation and culture of primary mouse hepatocytes by performing a two-step collagenase perfusion technique and discuss their utilization for investigating protein metabolism. The liver of an adult mouse is sequentially perfused with ethylene glycol-bis tetraacetic acid (EGTA) and collagenase, followed by the isolation of hepatocytes with the density gradient buffer. These isolated hepatocytes are viable on culture plates and maintain the majority of endowed characteristics of hepatocytes. These hepatocytes can be used for assessments of protein metabolism including nascent protein synthesis with non-radioactive reagents. We show that the isolated hepatocytes are readily controlled and comprise a higher quality and volume stability of protein synthesis linked to energy metabolism by utilizing the chemo-selective ligation reaction with a Tetramethylrhodamine (TAMRA) protein detection method and western blotting analyses. Therefore, this method is valuable for investigating hepatic nascent protein synthesis linked to energy homeostasis. The following protocol outlines the materials and methods for the isolation of high-quality primary mouse hepatocytes and detection of nascent protein synthesis.

Here, we present a protocol for the isolation of healthy and functional primary mouse hepatocytes. Instructions for detecting hepatic nascent protein synthesis by non-radioactive labeling substrate were provided to help understand the mechanisms underlying protein synthesis in the context of energy-metabolism homeostasis in the liver.

非放射性 L azidohomoalanine ラベル法による新生タンパク質合成解析用プライマリ マウス肝細胞の分離

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for ...

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

Mitochondria are essential organelles for the cellular metabolism and survival. A variety of key events take place in mitochondria, such as cellular respiration, oxidative metabolism, signal transduction, and apoptosis. Consequently, mitochondrial dysfunction is reported to play an important role in the antifungal drug tolerance and virulence of pathogenic fungi. Recent data have also led to the recognition of the importance of the mitochondria as an important contributor to fungal pathogenesis. Despite the importance of the mitochondria in fungal biology, standardized methods to understand its function are poorly developed. Here, we present a procedure to study the basal oxygen consumption rate (OCR), a measure of mitochondrial respiration, and extracellular acidification rates (ECAR), a measure of glycolytic function in C. albicans strains. The method described herein can be applied to any Candidaspp. strains without the need to purify mitochondria from the intact fungal cells. Furthermore, this protocol can also be customized to screen for inhibitors of mitochondrial function in C. albicans strains.

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

Here, we present a stepwise protocol to investigate the mitochondrial respiration and glycolytic function in Candida Albicans using an extra flux analyzer.

カンジダを使用してリアルタイム細胞フラックス解析のバイオ エネルギー調査

Mitochondria are essential organelles for the cellular metabolism and survival. A variety of key events take place in mitochondria, such as cellular ...