この研究は、CounterACTは、プログラム、国立衛生研究所（NIH）、ディレクターのOffice、および環境健康科学研究所（NIEHS）助成金番号U54 ES015678（CWW）でサポートされています。 SAはまた、小児病院コロラド州/コロラドスクールオブマインズコラボレーションパイロット賞＃G0100394および小児病院コロラド州リサーチインスティチュートパイロット賞＃のG0100471によってサポートされています。

1。ラット心筋細胞培養<ol><li>全ての実験は、施設内動物のケアと使用委員会、動物実験委員会によって承認されたプロトコルの下で実施された。 </li><li>以前に4記載の方法を使用してオスのラットの心臓（心室）（240〜260グラム）から、ラットの心筋細胞を得る。 （100 mg / kgで、つま先のピンチ方式による麻酔を確認する）簡単に言えば、ペントバルビタールの腹腔内注射を使用して動物を麻酔した後、10.0ミリリットル、クレブスリンガー緩衝液（pH7.4）を含む1のCa 2 +に心を取り除く。 </li><li>血液を除去した後、0.02％プロテアーゼおよび0.06％コラゲナーゼA（5.0ミリリットル/ハート）を含むCa 2 +を無料でクレブスリンガーバッファに切り替えることが心の5-6Xを洗浄します。時々振盪しながら37℃で10〜15分間この溶液に心をインキュベートする。 </li><li> 10〜15分後、さらに5分間のCa 2 +を含まないクレブスリンガー緩衝液で酵素液を洗い流す。弛緩性組織私たちから細胞を放出サスペンション上下に数回ピペッティングして25ミリリットルピペットをING。 </li><li> 70μmのナイロンメッシュを通して濾過することにより、組織から細胞を分離し、それらを0.1のCa 2 +を含むクレブスリンガー緩衝に定住することができます。 0.2のCa 2 +を含む1.0ミリリットルクレブスリンガー緩衝液中の細胞ペレットを一時停止します。 </li><li>慎重にnonmyocytesから心筋細胞を分離し、それらを30分間沈降させるために、15ミリリットルの遠心管の過5.0ミリリットル60μg/ mlのウシ血清アルブミン、BSAを、レイヤー。心筋細胞は重く、管の底部に移動する。慎重に上清を細胞および培地を除去します。さらに細胞を精製するために、一度、この手順を繰り返します。 </li><li>徐々に（0.25ミリモル、0.5ミリモル、0.75ミリモル、および緩衝液を含む1.0のCa 2 +を用いて段階的に）を1.0mMのCa 2 +緩衝液を含有するダルベッコ改変イーグル培地からなるACCT培地中に再懸濁する心室筋細胞/心筋細胞を洗浄することにより遷移DMEM、containingの2 mg / mlのBSA、2mMのL-カルニチン、5mMのクレアチン、5mMのタウリン、100 IU / mlペニシリン、および100μg/ mlのストレプトマイシン。 </li><li>プレート100ミリメートルまたは35ミリメートルのラミニンコーティングされたプラスチック製の培養皿またはラミニン（個/ cm 21μg）をでプレコート40×22ミリメートルのガラス製カバースリップ上/ mm 2の 100から150個の細胞の密度でACCT培地中で心筋細胞。 1時間後、接続されていない細胞を除去するために2.0ミリリットルACCTで皿を洗う。新鮮な培地の10.0ミリリットルを加え、細胞を培養する。 </li></ol>ヒト気道上皮基底細胞の2。差別気液界面（ALI）文化<ol><li>治験審査委員会の下に国民病研究インターチェンジからのヒト気管気管支組織を調達するには、プロトコルを承認した。細胞回収および公開された手順5,6を用いて培養を行う。彼らはTICをヒトの吸入曝露の正確なモデルであるため、この手順では、細胞培養を使用していますが、必要に応じて1アリMOで隔離し、成長することができます7,8を使用および/ ​​またはラットの気道上皮細胞。 </li><li>簡単に言えば、すべての結合組織とリンパ節を除去した後の組織の小片（〜¼インチ）を作る。乳酸リンゲル液中に組織を数回洗浄します。 50ミリリットルコニカルチューブに組織を追加し、（最小必須培地、MEM、流体比（1:10）に対する組織の1％Protease/0.01％のDNA分解酵素）プロテアーゼ溶液を加える。 </li><li>一晩4℃で組織を揺する。 10％ウシ胎児血清を添加することにより、組織の解離を終了する。外科用メスで上皮表面をこすり、遠心分離（10分間2,000 RPM）で細胞を回収。 </li><li>フルチャーら 9により詳細に記載されるように特別なALI培地中のコラーゲンコートスナップウェルに通路一方に板細胞を調製。頂端メディアを削除することによって気液界面（ALI）に変更する前に、5日間培養し。 </li><li> （隔日ALI培地を用いて細胞を供給し、細胞をさらに2〜3週間の分化を可能にするエクスポージャーを実行する前に、数々の暴行繊毛や粘液分泌）を観察。メディアの変化と一緒に温かいPBSで頂端面を洗浄します。 </li></ol> 3。塩素暴露<ol><li>偶然の塩素が漏れた場合に人員の露出を防止するために必要な二次封じ込めを提供100 FPMの運用面速度で修飾化学フード内の塩素露光システム（CES）が含まれている。 </li><li>若干の正の圧力（水の0.5インチ）の下でシステムを運用しています。乾燥空気は、15 L /分で供給される塩素の適切なレベルは、所望の最終塩素濃度を達成するために供給される。チャンバーは4ミニBCUロックと圧力シールを提供するために、低デュロメーターシリコーンガスケットと、ロック蓋を有する。 </li><li>マスフローコントローラーを介してCl 2の混合物を提供します。 CESは乾燥窒素中のCl 2 1.0％を含むガスボンベを使用しています。 </li><li>使用した希釈空気の流れを調節するカスタムコントロールパネルを設計し、同様に露光チャンバに送達Cl 2中の濃度を調節する。低容量サンプリングポンプは、次いで分析器に接続されたデータロガーに記録された濃度を監視するための塩素分析装置にチャンバからの排気を引く。 </li><li>露光前に等しく送達および排気速度を確保するために流量計を用いてチャンバ内の流速を測定する。 </li><li>上清培地を除去し、新鮮な培地（ALI培養における基底外側メディア）を追加することにより、露光のための細胞培養を準備します。薬との任意の前処理は、この時点で実行することができる。 </li><li> 2密封されたポリスルホン生物学的封じ込め室にCl 2ガス（30分50、100、または300 ppm）の気道上皮細胞のALI培養を公開します。心筋細胞（膜上の文化を水没やコンフルエント）を15分間50または100 ppmのClで2にさらされている。 </li><li>暴露後のCまで、空気で室内をフラッシュl 2はレベルが1ppm以下に低下し、安全に（5分以内）の細胞を除去するために開くことができる。 </li></ol> 4。経上皮電気抵抗（TER）測定<ol><li>気液界面のTER、アリ、塩化銀「箸」一対の電極を有する上皮voltohmmeterを使って文化を測定します。 </li><li>使用前に、ALIメディア15分で箸電極を平衡化させます。 </li><li>表面（2.0ミリリットル）（1.0ミリリットル）先端および側底に暖かいメディアを追加し、voltohmmeterの箸電極を用いてTERを測定します。 </li><li>頂端メディアにおける電極の短い腕と基底外側の媒体での長い腕を浸します。電気抵抗を評価するためにvoltohmmeterに「措置」ボタンをクリックしてください。 </li><li> voltohmmeterはオームまたはkのオームを測定するためのオプションがあります。トランスを得るために、各細胞層全体に測定された抵抗からの無細胞培養支持体間の抵抗を引く上皮抵抗（TER）。 </li></ol> 5。カスパーゼ測定<ol><li>基底外側表面の露光後に新鮮なALIメディア（2.0ミリリットル）を追加し、室温で細胞を培養する。 4および24時間後に上清メディアを収集します。 </li><li>商業カスパーゼ3/7アッセイキットを使用して、メディア上清中のカスパーゼ3/7活性を測定する。 </li></ol> 6。ウエスタンブロットおよび免疫細胞化学<ol><li>以前に10を説明したように細胞溶解物を用いたウエスタンブロットを実行します。 5×還元サンプル緩衝液中でタンパク質溶解物（20μgの）を一時停止し、5分間沸騰させる。 SDS-PAGE（4から15までパーセント）にタンパク質溶解物を対象とし、電気泳動ブロッティングによりニトロセルロース膜に分離したタンパク質を移す。 </li><li>洗浄緩衝液中の5％ミルク（PBS + 0.1％の界面活性剤）と共に膜をインキュベートすることによって非特異的結合をブロックし、4℃で一晩、1:1000希釈でSERCA2又はサルコメア·アクチンに対する一次抗体で膜をプローブ。次に、洗浄し、膜をインキュベートするそれぞれのペルオキシダーゼ結合二次抗体および市販のペルオキシダーゼ検出キットを用いて、10前に説明したように検出用に開発する。 </li><li>免疫細胞化学のためにPBSでリンス後20分間、10 mMクエン酸ナトリウム緩衝液中で0.4％トリトン-X-100で6ウェルプレートに挿入またはカバーガラス上で増殖させた生細胞を治療する。 </li><li> 20分間、5％ロバ血清で細胞を処理することにより非特異的結合ブロックし、次いで、非特異的IgG又はサルコメア·アクチン又はKi-67の特有の個々の一次抗体で細胞をインキュベートする。 </li><li> PBSで細胞を洗浄し、一次抗体および核染色（1μg/ mlのDAPI）を検出するために、蛍光結合二次抗体と共にインキュベートする。 </li><li> PBSで洗浄し、商業封入剤を使用したカバースリップをマウントします。 </li><li>蛍光顕微鏡を用いて染色を可視化する。 </li></ol>

<ol>
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The authors declare that they have no competing financial interests.

1が肺に有毒化学物質を呼吸する際に、急性毒性曝露の最も一般的なタイプが発生します。これらの化学物質はまた、迅速に血流中に取り込まれてもよく、例えば、脳や心臓などの他の臓器に影響を与えることができる。動物モデルを用いて種々の薬剤の吸入毒性が研究され、広く報告されている、しかし、メカニズムはあまりよく理解されている。これは効果的な治療法の開発に大きなハ?...

塩素などの有毒化学物質の吸入（TICを）/ガス（CL 2）への曝露は、偶発的暴露中だけでなく、化学的脅威エージェントとしての使用の可能性の継続的な健康問題のまま。肺が主なターゲットであるが、このような心臓や脳などの臓器はまた、in vivoモデルは、一般のTICからテストの毒性のために使用されています。1-3に影響を与えたが、毒性評価のためのin vitroアッセイで効果的なコスト、単純で、より速く、より公開されています。 でvitroモデルは、in vivoで評価することが困難な場合があるエージェント-細胞相互作用の広範な調査を可能にします。このようなインビトロ露光システムはまれであり、また、毒性因子は、細胞を浸漬した培養培地に添加されるいくつかの従来のモデルでは、薬剤の特性は、相互作用は、培地中の成分への結合を変更することができる。例えば、このようなシナリオの細胞培養系における気液界面（ALI）を直接ガス状物質にさらされる可能性がここで提案初代ヒト気道上皮細胞の培養物は有望であり得る。 気道の内側を覆う上皮細胞は、吸入毒性化学物質に対する防御の最初の行です。ヒト気道上皮は、肺内腔と基礎となる細胞との間の物理的な障壁を形成し、肺の応答に参加しています。これは、サイトカインおよび他のプロと抗炎症剤の数を生成だけでなく、粘液/上皮をカバーする、気道表面液（ASL）を分泌する。 インビトロ培養系に沈め従来における制限の1つは、上皮表面を覆うASL粘液を除去または希釈されることもある。これは、空気にさらされている肺上皮細胞の生理状態を反映していない。このように、TIC毒性試験のためのin vitro系における理想は、このアーキテクチャを複製する必要があります。迅速なスクリーニングmを開発することに大きな関心が寄せられている生体毒性を予測するethods。 ALIで増殖させた上皮細胞が水没し、気道の優れたモデルを提供し増殖した細胞と比較して、高分化型の構造と機能を差別化している。 本研究では、有毒吸入ガスの毒性をテストするためのヒト気道（気管支）上皮細胞の気液界面の文化の使用を記載し、したがって毒性のもう一つの重要な目標を検討し、心筋細胞の水没細胞培養と比較します。

<table border="0" cellpadding="0" cellspacing="0" width="1128">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Name</td>
			<td style="width:329px;">Company</td>
			<td style="width:147px;">Catalog Number</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Rats</td>
			<td style="width:329px;">Harlan Laboratories</td>
			<td>Sprague-Dawley&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Pentobarbital</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">P3761</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Chlorine</td>
			<td style="width:329px;">AirGas, Inc</td>
			<td style="width:147px;">X02NI99CP163LS1</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Caspase 3/7 kit&#160;</td>
			<td style="width:329px;">Promega</td>
			<td style="width:147px;">G8091</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Epithelial voltohmmeter and chopstick electrode</td>
			<td style="width:329px;">World Precision Instruments</td>
			<td style="width:147px;">EVOM and STX2</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Snapwell inserts</td>
			<td style="width:329px;">Corning</td>
			<td style="width:147px;">07-200-708</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">70 micron nylon cell strainer</td>
			<td style="width:329px;">Corning</td>
			<td style="width:147px;">#352360</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polysulfone biocontainment chambers&#160;</td>
			<td style="width:329px;">BCU, Allentown Cage Equipment</td>
			<td style="width:147px;">BCU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td style="width:329px;">Life technologies</td>
			<td style="width:147px;">12491-015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Sarcomeric actin antibody</td>
			<td style="width:329px;">Abcam Cambridge, MA</td>
			<td style="width:147px;">ab28052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SERCA2 antibody</td>
			<td style="width:329px;">&#160;Affinity Bioreagents, Golden, CO</td>
			<td style="width:147px;">MA3-9191</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ki-67 antibody</td>
			<td style="width:329px;">&#160;Dako, Carpinteria, CA</td>
			<td style="width:147px;">M7248</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa-488-conjugated secondary antibody</td>
			<td style="width:329px;">&#160;Invitrogen, Grand Island, NY</td>
			<td style="width:147px;">A11029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">BSA</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">A9418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Carnitine</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">C0283</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Taurine</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">T8691</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Creatinine</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">C6257</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Krebs Ringer Buffer</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">K4002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Protease</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">P5147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Collagenase</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">C6885</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; DNAase</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">DN-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Lactated Ringer solution</td>
			<td style="width:329px;">Abott Laboratories</td>
			<td style="width:147px;">7953</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Donkey serum</td>
			<td style="width:329px;">Fisher Scientific</td>
			<td style="width:147px;">017-000-001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">PBS, phosphate buffered saline</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">D1408</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">4-15% SDS-PAGE gels</td>
			<td style="width:329px;">Bio-Rad</td>
			<td style="width:147px;">456-1083</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Nitrocellulose membrane</td>
			<td style="width:329px;">Bio-Rad</td>
			<td style="width:147px;">162-0115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Dergent, Tween&#160;</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">P1379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Peroxidase detection kit</td>
			<td style="width:329px;">Pierce</td>
			<td align="right" style="width:147px;">3402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">DAPI</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">D9542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Mounting media, Fluormount G</td>
			<td style="width:329px;">eBiosciences</td>
			<td style="width:147px;">00-4958-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Sodium citrate</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">71497</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Collagen</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">C7521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">MEM</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">M8028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Laminin</td>
			<td style="width:329px;">BD biosciences</td>
			<td style="width:147px;">354259</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Penicillin/Streptomycin</td>
			<td style="width:329px;">Life Technologies</td>
			<td style="width:147px;">15070063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">FBS</td>
			<td style="width:329px;">Gibco</td>
			<td style="width:147px;">200-6140AJ</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro cell culture model for toxic inhaled chemical testing

細胞培養物は、従来の動物モデルにおけるそれらの使用、治療薬の効力を開発し研究することが不可欠である。我々は、よく分化したヒト気道上皮および心筋細胞をモデル化する独特の能力を有する。これは、通常、細胞表面と相互作用し、水と反応し、水没の文化でその効果を制限する際に、様々な副生成物を形成することができる塩素などの有毒化学物質の吸入、の有害な影響を研究するための貴重なツールである可能性があります。エアliqiuid界面における高分化ヒト気道上皮細胞培養物を使用して我々のモデルは、この制限を回避するだけでなく、潜在的な有毒化学物質の吸入毒性の重要なメカニズムを評価する機会を提供します。私たちは、このような塩素暴露などの有毒化学物質の吸入の際に強化された膜の完全性の損失、カスパーゼリリースと死について説明します。この記事では、哺乳動物の心臓や気道上皮Cの塩素暴露をモデル化する方法が提案されている文化とこれらの細胞型への影響を評価するための簡単​​なテストでのエル。

主な棒状の心筋細胞は、ラミニン行列やスプレッドに接続し、コンフルエントな培養物（ 図1Aおよびその挿入図）に分化する。これらの細胞はさらに筋節アクチンおよびSERCA2発現（ 図1Bおよび1C）に基づいて特徴づけた。ラットの心筋細胞は、ラミニンコーティングされた膜（ 図1D）上で増殖させた細胞に水没文化や融合性層の破壊で広範な...

Watch this Scientific Journal Video about In vitro Cell Culture Model for Toxic Inhaled Chemical Testing at JoVE.com

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

このプロトコルは、有毒な化学物質を吸入するための細胞培養物の露光方法を実証するために設計されている。差別化され気液界面の気道上皮細胞の（ALI）の文化の暴露は、塩素などの有毒ガスに対する気道曝露のユニークなモデルを提供します。本稿では、上皮細胞および心筋細胞の液体培養の気液界面培養物における塩素曝露の影響を記載している。 インビトロ露光システムは、重要な機構的研究は、次いで、新規な治療薬を開発するために利用することができる経路を評価することを可能にする。

有毒吸入化学試験のためのin vitroでの細胞培養モデルにおける

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to ...

in-vitro-cell-culture-model-for-toxic-inhaled-chemical-testing

Research

JoVE Journal

Bioengineering

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

10.6K ビュー.  University of Colorado. このプロトコルは、有毒な化学物質を吸入するための細胞培養物の露光方法を実証するために設計されている。差別化され気液界面の気道上皮細胞の（ALI）の文化の暴露は、塩素などの有毒ガスに対する気道曝露のユニークなモデルを提供します。本稿では、上皮細胞および心筋細胞の液体培養の気液界面培養物における塩素曝露の影響を記載している?...

ビデオ: In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

腫瘍微小環境を再作成するためのマイクロ流体デバイス<em&gt;体外で</em

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

生物工学

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape.
Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture.
Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

アクティブな細胞培養のための形状記憶ポリマー

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk of thromboemboli formation1,2,3. We have developed a method to line the inside surface of Ti tubes with autologous blood-derived human or porcine endothelial progenitor cells (EPCs)4. By implanting Ti tubes containing a confluent layer of porcine EPCs in the inferior vena cava (IVC) of pigs, we tested the improved biocompatibility of the cell-seeded surface in the prothrombotic environment of a large animal model and compared it to unmodified bare metal surfaces5,6,7 (Figure 1). This method can be used to endothelialize devices within minutes of implantation and test their antithrombotic function in vivo. 
Peripheral blood was obtained from 50 kg Yorkshire swine and its mononuclear cell fraction cultured to isolate EPCs4,8. Ti tubes (9.4 mm ID) were pre-cut into three 4.5 cm longitudinal sections and reassembled with heat-shrink tubing. A seeding device was built, which allows for slow rotation of the Ti tubes. 
We performed a laparotomy on the pigs and externalized the intestine and urinary bladder. Sharp and blunt dissection was used to skeletonize the IVC from its bifurcation distal to the right renal artery proximal. The Ti tubes were then filled with fluorescently-labeled autologous EPC suspension and rotated at 10 RPH x 30 min to achieve uniform cell-coating9. After administration of 100 USP/ kg heparin, both ends of the IVC and a lumbar vein were clamped. A 4 cm veinotomy was performed and the device inserted and filled with phosphate-buffered saline. As the veinotomy was closed with a 4-0 Prolene running suture, one clamp was removed to de-air the IVC. At the end of the procedure, the fascia was approximated with 0-PDS (polydioxanone suture), the subcutaneous space closed with 2-0 Vicryl and the skin stapled closed. 
After 3 - 21 days, pigs were euthanized, the device explanted en-block and fixed. The Ti tubes were disassembled and the inner surfaces imaged with a fluorescent microscope.
We found that the bare metal Ti tubes fully occluded whereas the EPC-seeded tubes remained patent. Further, we were able to demonstrate a confluent layer of EPCs on the inside blood-contacting surface.
Concluding, our technology can be used to endothelialize Ti tubes within minutes of implantation with autologous EPCs to prevent thrombosis of the device. Our surgical method allows for testing the improved biocompatibility of such modified devices with minimal blood loss and EPC-seeded surface disruption.

A method for seeding titanium blood-contacting biomaterials with autologous cells and testing biocompatibility is described. This method uses endothelial progenitor cells and titanium tubes, seeded within minutes of surgical implantation into porcine venae cavae. This technique is adaptable to many other implantable biomedical devices.

大動物モデルにおける心血管デバイス用自家血管内皮前駆細胞播種技術と生体適合性試験

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk ...

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

ヒト細胞培養のための回転細胞培養システム：モデルとしてのヒト栄養膜細胞

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

再構成ヒト角膜上皮様（RHCE）組織モデルを使用して眼刺激性化学物質の有害性の同定のための眼刺激性試験（EIT）

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

細胞培養アプリケーションのためのヒドロゲルの光媒介形成とパターニング

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

アン<em&gt;インビトロ</emマウス椎間板の&gt;器官培養モデル

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are an emerging treatment for photoreceptor degenerative diseases that seek to restore vision by artificially stimulating the surviving retinal neurons in the hope of eliciting comprehensible visual perception in patients. Current retinal prostheses have demonstrated success in restoring limited vision to patients using an array of electrodes to electrically stimulate the retina but face substantial physical barriers in restoring high acuity, natural vision to patients. Chemical neurostimulation using native neurotransmitters is a biomimetic alternative to electrical stimulation and could bypass the fundamental limitations associated with retinal prostheses using electrical neurostimulation. Specifically, chemical neurostimulation has the potential to restore more natural vision with comparable or better visual acuities to patients by injecting very small quantities of neurotransmitters, the same natural agents of communication used by retinal chemical synapses, at much finer resolution than current electrical prostheses. However, as a relatively unexplored stimulation paradigm, there is no established protocol for achieving chemical stimulation of the retina in vitro. The purpose of this work is to provide a detailed framework for accomplishing chemical stimulation of the retina for investigators who wish to study the potential of chemical neuromodulation of the retina or similar neural tissues in vitro. In this work, we describe the experimental setup and methodology for eliciting retinal ganglion cell (RGC) spike responses similar to visual light responses in wild-type and photoreceptor-degenerated wholemount rat retinas by injecting controlled volumes of the neurotransmitter glutamate into the subretinal space using glass micropipettes and a custom multiport microfluidic device. This methodology and protocol are general enough to be adapted for neuromodulation using other neurotransmitters or even other neural tissues.

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

This protocol describes a novel method for investigating a form of chemical neurostimulation of wholemount rat retinas in vitro with the neurotransmitter glutamate. Chemical neurostimulation is a promising alternative to the conventional electrical neurostimulation of retinal neurons for treating irreversible blindness caused by photoreceptor degenerative diseases.

バイオミメティック化学ニューロモデュレーション神経伝達物質グルタミン酸の in Vitroラット網膜のための方法論

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

マトリックス細胞代謝クロストークを研究するためのヒト3D細胞外マトリックス-脂肪細胞培養モデル

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

In Vitro Model of Human Cutaneous Hypertrophic Scarring using Macromolecular Crowding

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol applies a macromolecular crowding (MMC) technique to mimic this physiological crowding through the addition of neutral polymers (crowders) to cell cultures in vitro. Previous studies using Ficoll or dextran as crowders demonstrate that the expression of collagen I and fibronectin in WI38 and WS-1 cell lines are significantly enhanced using the MMC technique. However, this technique has not been validated in primary hypertrophic scar-derived human skin fibroblasts (hHSFs). As hypertrophic scarring arises from the excessive deposition of collagen, this protocol aims to construct a collagen-rich in vitro hypertrophic scar model by applying the MMC technique with hHSFs. This optimized MMC model has been shown to possess more similarities with in vivo scar tissue compared to traditional 2-dimensional (2-D) cell culture systems. In addition, it is cost-effective, time-efficient, and ethically desirable compared to animal models. Therefore, the optimized model reported here offers an advanced &#8220;in vivo-like&#8221; model for hypertrophic scar-related studies.

This protocol describes the use of macromolecular crowding to create an in vitro human hypertrophic scar tissue model that resembles in vivo conditions. When cultivated in a crowded macromolecular environment, human skin fibroblasts exhibit phenotypes, biochemistry, physiology, and functional characteristics resembling scar tissue.

高分子群集を用いたヒト皮萎縮性瘢痕化のインビトロモデル

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol ...