これらのプロトコルの多くの改善への貢献のために明ミン·リー、キャスリンPlichta、ビクトリアミランダ·トンプソンとサム·キュッキム：著者は、在庫ラボの過去のメンバーに感謝したい。この作品は、SP株に国立科学財団の助成金IOS-0840932およびIOS-0724978によって部分的に資金を供給された

彼らのSymboitic細菌と昆虫病原線虫の1。 インビボ飼育<ol><li> 100×15ミリメートルのプラスチックシャーレを反転し、皿のふたにろ紙（90ミリメートル）の2つのディスクを配置します。 </li><li>均等に濾紙上（1,000〜2,000 IJ / mlの濃度で）IJ（感染少年）水懸濁液1mlを分配する。 注：IJSは表面殺菌する必要はありません。 </li><li>皿に大きなwaxmothのハチノスツヅリガの10終齢幼虫を追加します。目標は、約100〜200 IJS /幼虫を提供することにある。 </li><li>ペトリ皿（ 図1）の底部とふたをカバーし、ペトリ皿にラベルを付ける。以下の情報に言及：線虫種名（既知の場合）;コー​​ド/指定を隔離する。日付感染トラップが設定されました。 </li><li> （水分を節約するために）緩く密封されたビニール袋の中に皿を置き、どちら室温または20〜25℃の間のインキュベーター内で暗所に保管してください。毎日の料理をチェックする 注：それは土壌中に自然感染状態を模倣として闇は、線虫の感染を容易にします。 </li><li> 3〜5日後に、変更されたホワイトトラップ7への線虫感染の兆候で死体を取り除く。 注：死体が腐敗の臭い場合、これは培養が成功しなかった、および/または汚染があったことを示す可能性があります。線虫および昆虫の新しいバッチで新しい感染室を設定します。 </li></ol>彼らの共生細菌を持つ昆虫病原線虫の2 のin vitro飼育<ol><li>肝臓、腎臓天法9,19 <ol><li>小片（2 cm 3程度以下）に肝臓と腎臓をみじん切りにし、NaCl、寒天と水300mlでブレンダーに入れ、肉が液体パルプ、濃厚なペーストやピューレになるまでブレンドする。その後、1 Lの三角フラスコに移し、混合物。 </li><li>ブレンダー容器に水の最終200ミリリットルを加え、中に残っているメディアをデカント三角フラスコ。 121°Cで15分間オートクレーブ。許可オートクレーブ後の寒天注ぐ前に触れて冷却する。 注：この培地は、肉の塊を持っている傾向があるため、媒体のピペッティングは推奨されません。 </li><li>手の攪拌または間の三角フラスコを旋回がより均質なメディアに注ぐながら5（または6）cmのペトリ皿に寒天〜20ミリリットルを注ぐ。必要になるまで寒天は4℃で固化し、店の食器を許可する 注：ペトリ皿に注ぎ、肉のいずれかの大きな塊を破壊する火炎滅菌金属スパチュラを使用してください。 </li></ol></li><li>脂質天法9,19 <ol><li>栄養ブロス、寒天及び酵母エキスを一緒に混合することにより、脂質寒天培地を準備し、2リットルの三角フラスコに混ぜ注ぐ。 121℃で15分間、H 2 OおよびMgCl 2•6H 2 O、オートクレーブ蒸留追加。 </li><li>コー​​ン油、コーンシロップミックスの無菌ミックスを加え、攪拌または均等に油滴を分散させるために積極的かつ頻繁に渦巻く。 <br/&gt;注：UVクロスリンカーに必要な容積を配置することによって、コーン油及びシロップを滅菌する。オイルが液体に溶解するが、それは小さな液滴中にあることを確認することはありません。 </li><li> 5（または6）cmのペトリ皿上に〜20ミリリットル寒天を注ぎ、必要になるまで寒天4℃で固化し、店の料理をすることができます。 </li><li>ストリークは、脂質寒天プレートに共生細菌を希望し、24〜48時間、28℃でプレートをインキュベートする。 注：一晩（12-16時間）のLBサブカルチャーの100〜200μlのスプレッド。 </li><li>翌日は、約0.4ミリリットル表面殺菌し、線虫懸濁液（卵や感染少年）のを追加し、暗いまたは22±3℃のインキュベーター内で、室温でプレートをインキュベートする。 注：それは、線虫の成長に好ましくない過度に湿潤環境を作成することができますように線虫懸濁液を超える0.4ミリリットルを追加しないでください。 </li><li>毎日文化を監視します。培養物は、約12日間（ 図2）にIJSの新世代を生成する必要があります。</li><li>線虫が変更ホワイトトラップにディッシュ（ 図3A）の側面をクロール見ているときIJS（ 図3B）を収穫する（オロスコら 12参照）寒天でボトムディッシュを転送します。メディアの汚染を減らすために、層流フードの下に、この手順を実行します。 </li><li>出現時に変更されたホワイトトラップからIJSを収集し、あらゆる媒体破片を除去するIJサスペンションをすすぐ。ストア（コールド温度インキュベーターまたは低温室）で10℃での滅菌蒸留水にIJまたは必要に応じてすぐに使用しています。 注：研究の目的に応じて、シンビオント·保菌またはaposymbiotic線虫のいずれかでプレートをシード。 </li></ol></li></ol> 3 インビトロ Aposymbioticの飼育（共生フリー）昆虫病原線虫<ol><li> 10-20 G.に感染希望線虫種のIJSとmellonella幼虫 （セクション1を参照）。 3または4日後（この時点でIJSは成熟しているはず第一世代の成人に）死体を収集します。 注：卵の適切な数を得るために必要な雌の成熟度と数に時間が種によって異なります。より多くのGを感染させるmellonella幼虫必要であれば、彼らは右の発達段階にあるかどうかを評価するために、早ければ48時間後に感染症などの解剖を開始します。 </li><li>生理食塩水（M9バッファ18）とのペトリ皿に個別に死体を置きます。先端の細い鉗子で頭を引いて、死体を解剖。 </li><li>細い針（好ましくはL字型）で、少なくとも20（内側卵）妊娠、女性を集め、M9バッファー（ 図4A）の10ミリリットルを含む時計皿に置いてください。 </li><li>以下の成分を考慮してaxenizing溶液を調製：蒸留水6.75ミリリットル、1.25ミリリットル5NのNaOH、および2.25ミリリットルの市販の漂白剤溶液を3％の次亜塩素酸を希釈した。 注：NaOHを塩基性溶液、手袋、ゴーグルや他の適切な保護である衣服は着用してください。 注：漂白剤は、光の中で分解する、新鮮なaxenizing液を毎回準備します。 axenizing液のいくつかのバッチを用意し、複数の時計皿に卵の表面殺菌や収穫を行う。 </li><li> M9バッファは緩衝液で時計皿を充填し、ピペットを用いてバッファを吸い取って、女性には少なくとも3回洗浄します。女性は時計皿に残っていることを確認します。このステップをさらに2回繰り返します。 </li><li>すぐにaxenizingソリューションを追加し、女性は10〜15分を超えないために、この溶液中に残存することができます。 （axenizing液に抵抗性である）卵は無傷のままでいる間に崩壊し始めて線虫の組織を観察します。 </li><li>手動で針またはメス（ 図4B、C）でそれらを切断することによって、プロセスをスピードアップするために女性の身体を破壊する。未溶解組織を加える避け、1.5mlのマイクロチューブにそれらをピペッティングすることにより、卵を除去します。 注：Mとして使用卵を収集し、卵の生存能力として、迅速に作業するために、必要に応じて、任意のマイクロチューブはaxenizing溶液への曝露の長期間にわたって減少します。 </li><li> 「高速設定」を使用して15秒間ボルテックスチューブは、更に、卵に付着線虫組織を除去した。別の方法として、渦が利用できない場合、解決策を上下にピペットを数回。 </li><li>で約18000グラムで2分間の遠心分離によってペレット卵。卵は十分にペレットされない場合は速度を上げてください。 axenizing液を取り出し、1分間900×gで滅菌蒸留水と遠心機1のより多くの時間をマイクロチューブに充填することにより二回すすいでください。 </li><li>最後の洗浄後、滅菌蒸留水300〜500μlの卵を再懸濁し、滅菌3センチメートルペトリ皿または滅菌時計皿に置いてください。卵は今表面殺菌であることを忘れないでください、そう卵の清浄度を維持するために、滅菌ピペットおよび/またはpippetterのヒントや水を使用しています。 </li><li>彼らは（ 図4D）は無傷であることを確認するために解剖顕微鏡（30-50X倍率）の下で卵を調べ、肝臓、腎臓寒天と5cmのプレートに移す（セクション2.1を参照） 注：それは寒天が過度に濡れになりますので、プレート上の卵の懸濁液の400以上のμLを追加しないでください。 </li><li>適切な情報をラベルプレート、28℃の暗所で直立に格納します。卵の孵化することは明白で、一晩でなければなりません。 注：水は皿の蓋の上に凝縮することがありますが、それは1日または2日後に蒸発する。この時、寒天の水分を保持し、その乾燥を避けるために、ビニール袋に皿を置く。 </li><li>定期的に料理を守り、真菌や細菌汚染の兆候を示すあらゆる料理を捨てる。 IJSがペトリ皿の壁を移行見たら、ふたを取り外して、修正されたホワイトトラップ12に寒天でボトムディッシュを転送します。 modifieに皿を転送する際に、無菌状態を考慮してくださいdはホワイトトラップが露出メディアの汚染を回避する。 </li><li>必要に応じてホワイトトラップと収穫IJの​​監視を続けます。 注：Aposymbiotic線虫は何日あっても数週間ホワイトトラップの水の中に移動していきます。 </li></ol>

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利害関係が宣言されていない。

適当な宿主を使用すると、EPNの生体内で飼育することに成功するための重要な要因である。通常、両方steinernematidsとheterorhabditidsを再現することができますし、正常に高いワックス蛾、 ハチノスツヅリガ （鱗翅目：メイガ）の幼虫で、そのライフサイクルを完了。しかし、異なるファミリーおよび/または指図から他の昆虫種が考えられる。現在記載され線虫種のいくつか...

昆虫病原性線虫（EPN）SteinernemaとHeterorhabditis属 。 （Steinernematidae、Heterorhabditidae）とそれらの細菌の共生、XenorhabdusとのPhotorhabdus属（腸内細菌）が陸生動物-微生物共生関係2-4,6,10,19。XenorhabdusとのPhotorhabdus属の創発モデルと考えられている。また、感染性若年性（IJ）として知られている線虫の唯一の自由生活段階、または第3段階感染幼若8,10,13の腸内に共生として保有されている。細菌線虫ペアは昆虫の広い範囲の病原性であり、成功裏に6,8、世界中の生物学的制御と統合された害虫管理プログラムに実装されています。 ここで私たちは、生体内で 、頻繁に実験室条件下でEPNの飼育のために行使されたインビトロ技術での選択を示している。 私をnは、生体内の方法は、線虫の飼育用の昆虫宿主を企図している。通常、さまざまな昆虫の受注（ すなわち鱗翅目 、 甲虫目 、 双翅目 、 等 ）の未成熟な段階は、適切なホストとみなされます。メソッドは、通常、実験室での線虫の培養物の維持のために考慮されるインビボ 。線虫の量産を考慮した場合、この方法は適切でないかもしれない。昆虫宿主の大量のは、より多くの時間と昆虫飼育に関連する追加費用を要求して、この目的のために必要な場合があります。 昆虫病原性線虫には、いくつかのメディアに対してインビトロで培養することができる。研究の目的に応じて、 インビトロ法で 、またはメディア内共生細菌の混入を考慮することができる。この発表では、EPNの伝播のための2つの一般的に使用される方法が記載されている。培地の成分は、共生細菌aの栄養素の供給源を提供ND線虫用のステロール源。 インビトロ法は、昆虫宿主なしで優位にEPNの飼育を提供します。 適切な昆虫宿主が利用できない場合、もともと開発されたインビトロでのメディアの多くは、EPNの乗算のために使用した。しかし、過去数年間にわたり、in vitroでの飼育方法が広くEPNとその共生細菌17,19間の共生関係を理解することを目指して研究に採用になっている。 このプレゼンテーションで詳述技術は、さまざまな著者によって記載され、証券研究所、アリゾナ大学（ツーソン、アリゾナ州、米国）によって洗練ものに対応する。これらの技術は、害虫管理の目的のためにこれらの生物の大量生産に使用される技法の本体から区別される。

<table border="0" cellpadding="0" cellspacing="0" width="1827">
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		<tr height="42">
			<td height="42" style="height:42px;width:485px;">Material&#160;</td>
			<td style="width:260px;">Company</td>
			<td style="width:488px;">Catalog Number</td>
			<td style="width:595px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;"></td>
			<td style="width:260px;"></td>
			<td style="width:488px;"></td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:485px;">For in vivo Infections</td>
			<td style="width:260px;"></td>
			<td style="width:488px;"></td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:485px;">100 x 15 Petri Dishes</td>
			<td style="width:260px;">VWR</td>
			<td style="width:488px;">25384-088</td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:485px;">Filter Paper, 9 cm Grade 1 Cellulose</td>
			<td style="width:260px;">Whatman/VWR</td>
			<td style="width:488px;">28450-081</td>
			<td style="width:595px;">Grade 1 filter paper recommended</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:485px;">Insect hosts</td>
			<td style="width:260px;">Timberline</td>
			<td style="width:488px;">http://www.timberlinefisheries.com/ProductDetails.asp?ProductCode=WAXLG</td>
			<td style="width:595px;">Galleria mellonella are recommended</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;"></td>
			<td style="width:260px;"></td>
			<td style="width:488px;"></td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:485px;">For Liver-Kidney Agar (for 500 ml)</td>
			<td style="width:260px;"></td>
			<td style="width:488px;"></td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;width:485px;">60 x 15 mm Petri Dishes</td>
			<td style="width:260px;">VWR</td>
			<td>25384-092</td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:485px;">Beef Liver, 50 g</td>
			<td style="width:260px;">Locally sourced; butcher or supermarket</td>
			<td style="width:488px;"></td>
			<td style="width:595px;">Remaining may be stored frozen and thawed for future use</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:485px;">Beef Kidney, 50 g</td>
			<td style="width:260px;">Locally sourced; butcher or supermarket</td>
			<td style="width:488px;"></td>
			<td style="width:595px;">Remaining may be stored frozen and thawed for future use</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:485px;">Sodium Chloride 2.5g ( 0.5% final concentration )</td>
			<td style="width:260px;">Acros/VWR</td>
			<td>200002-434</td>
			<td style="width:595px;">any research grade NaCl can be used</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:485px;">Agar, 7.5 g (1.5% agar, final concentration)</td>
			<td style="width:260px;">HiMedia/VWR</td>
			<td>95026-642</td>
			<td style="width:595px;">any media grade agar can be used</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">500 ml distilled H20&#160;</td>
			<td style="width:260px;"></td>
			<td style="width:488px;"></td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:260px;"></td>
			<td style="width:488px;"></td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:485px;">For Lipid Agar (for 1 L)</td>
			<td style="width:260px;"></td>
			<td style="width:488px;"></td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:485px;">100 x 15 Petri Dishes</td>
			<td style="width:260px;">VWR</td>
			<td>25384-088</td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:485px;">Nutrient Broth, 8 g</td>
			<td style="width:260px;">BD/VWR</td>
			<td>90002-660</td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:485px;">Yeast Extract, 5g</td>
			<td style="width:260px;">EMD/VWR</td>
			<td>EM1.03753.0500</td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:485px;">Magnesium Chloride Hexahydrate,&#160; 10 ml (0.2g/ml)</td>
			<td style="width:260px;">EMD/VWR</td>
			<td>EM-MX0045-1</td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:485px;">Corn Oil, 4 ml</td>
			<td style="width:260px;">Any brand</td>
			<td style="width:488px;">Locally source; supermarket</td>
			<td style="width:595px;">Any brand of corn oil can be used</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:485px;">Corn Syrup, 96 ml combine 7 ml corn syrup in 89 ml heated H20 and swirl for homogeneity</td>
			<td style="width:260px;">Karo</td>
			<td style="width:488px;">Locally sourced; supermarket</td>
			<td style="width:595px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agar, 15 g</td>
			<td style="width:260px;">HiMedia/VWR</td>
			<td>95026-642</td>
			<td style="width:595px;">any media grade agar can be used</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Distilled H20,890 ml</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo and in vitro rearing of entomopathogenic nematodes (steinernematidae and heterorhabditidae)

昆虫病原性線虫（EPN）（SteinernematidaeとHeterorhabditidae）は 、腸内細菌科にグラム陰性ガンマプロテオバクテリアと共生のパートナーシップを持っている。 フォトルハブダスは heterorhabditidsの共生している間Xenorhabdus細菌がsteinernematids線虫に関連付けられています。一緒線虫や細菌は親密で、特定の提携で昆虫種の広い範囲を殺す強力な殺虫複合体を形成する。ここで、私たちは、生体内で 、一般に実験室条件下で、これらの線虫の飼育に使用されるインビトロ技術で実証している。さらに、これらの技術は、EPN培養の確立に成功するための重要なステップを表し、また、研究のために、これらの生物を利用する他のバイオアッセイの基礎を形成する。 aposymbiotic（シンビオントフリー）線虫の生産は、多くの場合、深いと共生の研究に多面的なアプローチのために重要である。このプロトコルは、抗生物質の添加を必要とせず、標準的な実験装置を用いて短時間で行うことができる。貯蔵におけるそれらの生存が使用される種に依存して変化し得るが、このようにして製造線虫は、比較的ロバストである。このプレゼンテーションで詳述技術は、P株価の研究所、アリゾナ大学（ツーソン、アリゾナ州、米国）により、さまざまな著者によって記載され、洗練されたものに対応する。これらの技術は、害虫管理の目的のためにこれらの生物の大量生産に使用される技法の本体から区別される。

in vivoでの飼育方法は、線虫の成長と再生のための宿主としてのライブ昆虫を使用しています。感染室は昆虫のIJSを露出させるための効率的な方法である。これは別の昆虫宿主から細菌共生ベクトル線虫のライフサイクルにおける唯一のステージである。1は感染室だけでなく、このチャンバーを構築するために必要な材料のために設定示しています。 イン?...

Watch this Scientific Journal Video about In vivo and In vitro Rearing of Entomopathogenic Nematodes Steinernematidae and Heterorhabditidae at JoVE.com

In vivo and In vitro Rearing of Entomopathogenic Nematodes Steinernematidae and Heterorhabditidae

このプレゼンテーションの目的は、 生体内および昆虫病原性線虫の飼育のためのインビトロ技術に実証することである。 インビトロ法は、リッチ寒天培地を利用し、一方、 インビボの方法は、昆虫宿主でこれらの線虫の飼育を考えてみましょう。

in vivoおよび昆虫病原線虫の体外飼育には（SteinernematidaeとHeterorhabditidae）

Entomopathogenic nematodes (EPN) (Steinernematidae and Heterorhabditidae) have a mutualistic partnership with Gram-negative Gamma-Proteobacteria in the ...

in-vivo-vitro-rearing-entomopathogenic-nematodes-steinernematidae

Research

JoVE Journal

Bioengineering

In vivo and In vitro Rearing of Entomopathogenic Nematodes (Steinernematidae and Heterorhabditidae)

20.4K ビュー.  University of Arizona. このプレゼンテーションの目的は、 生体内および昆虫病原性線虫の飼育のためのインビトロ技術に実証することである。 インビトロ法は、リッチ寒天培地を利用し、一方、 インビボの方法は、昆虫宿主でこれらの線虫の飼育を考えてみましょう。 

ビデオ: In vivo and In vitro Rearing of Entomopathogenic Nematodes Steinernematidae and Heterorhabditidae

Decellularization and Recellularization of Whole Livers

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to recreate or mimic the liver microstructure with grounds up approach using tissue engineering and microfabrication techniques have not been successful so far due to this design challenge. In addition, synthetic biomaterials used to create scaffolds for liver tissue engineering applications have been limited in inducing tissue regeneration and repair in large part due to the lack of specific cell binding motifs that would induce the proper cell functions2. Decellularized native tissues such blood vessels3and skin4on the other hand have found many applications in tissue engineering, and have provided a practical solution to some of the challenges. The advantage of decellularized native matrix is that it retains, to an extent, the original composition, and the microstructure, hence enhancing cell attachment and reorganization5.
In this work we describe the methods to perform perfusion-decellularization of the liver, such that an intact liver bioscaffold that retains the structure of major blood vessels is obtained. Further, we describe methods to recellularize these bioscaffolds with adult primary hepatocytes, creating a liver graft that is functional in vitro, and has the vessel access necessary for transplantation in vivo.

Perfusion decellularization is a novel technique to produce whole liver scaffolds that retains the organ's extracellular matrix composition and microarchitecture. Herein, the method of preparing whole organ scaffolds using perfusion decellularization and subsequent repopulation with hepatocytes is described. Functional and transplantable liver grafts can be generated using this technique.

全肝臓の細胞化とRecellularization

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to ...

生物工学

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

タンパク質、細胞閉じ込めのための二次元加工基板を作成する

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Video-rate Scanning Confocal Microscopy and Microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical sectioning of complex systems. Confocal microscopy is routinely used, for example, to study specific cellular targets1, monitor dynamics in living cells2-4, and visualize the three dimensional evolution of entire organisms5,6. Extensions of confocal imaging systems, such as confocal microendoscopes, allow for high-resolution imaging in vivo7 and are currently being applied to disease imaging and diagnosis in clinical settings8,9.
Confocal microscopy provides three-dimensional resolution by creating so-called "optical sections" using straightforward geometrical optics. In a standard wide-field microscope, fluorescence generated from a sample is collected by an objective lens and relayed directly to a detector. While acceptable for imaging thin samples, thick samples become blurred by fluorescence generated above and below the objective focal plane. In contrast, confocal microscopy enables virtual, optical sectioning of samples, rejecting out-of-focus light to build high resolution three-dimensional representations of samples.
Confocal microscopes achieve this feat by using a confocal aperture in the detection beam path. The fluorescence collected from a sample by the objective is relayed back through the scanning mirrors and through the primary dichroic mirror, a mirror carefully selected to reflect shorter wavelengths such as the laser excitation beam while passing the longer, Stokes-shifted fluorescence emission. This long-wavelength fluorescence signal is then passed to a pair of lenses on either side of a pinhole that is positioned at a plane exactly conjugate with the focal plane of the objective lens. Photons collected from the focal volume of the object are collimated by the objective lens and are focused by the confocal lenses through the pinhole. Fluorescence generated above or below the focal plane will therefore not be collimated properly, and will not pass through the confocal pinhole1, creating an optical section in which only light from the microscope focus is visible. (Fig 1). Thus the pinhole effectively acts as a virtual aperture in the focal plane, confining the detected emission to only one limited spatial location.
Modern commercial confocal microscopes offer users fully automated operation, making formerly complex imaging procedures relatively straightforward and accessible. Despite the flexibility and power of these systems, commercial confocal microscopes are not well suited for all confocal imaging tasks, such as many in vivo imaging applications. Without the ability to create customized imaging systems to meet their needs, important experiments can remain out of reach to many scientists.
In this article, we provide a step-by-step method for the complete construction of a custom, video-rate confocal imaging system from basic components. The upright microscope will be constructed using a resonant galvanometric mirror to provide the fast scanning axis, while a standard speed resonant galvanometric mirror will scan the slow axis. To create a precise scanned beam in the objective lens focus, these mirrors will be positioned at the so-called telecentric planes using four relay lenses. Confocal detection will be accomplished using a standard, off-the-shelf photomultiplier tube (PMT), and the images will be captured and displayed using a Matrox framegrabber card and the included software.

The complete construction of a custom, real-time confocal scanning imaging system is described. This system, which can be readily used for video-rate microscopy and microendoscopy, allows for an array of imaging geometries and applications not accessible using standard commercial confocal systems, at a fraction of the cost.

ビデオレートでスキャニング共焦点顕微鏡とMicroendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical ...

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

マウスの心臓の活動電位およびカルシウムトランジェントの光学的マッピング

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

パラメータ化拡張期充満フォーマリズムを経由Transmitral流れの運動学的モデルベースの​​解析によるグローバル拡張期機能の定量化

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

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 ...

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 ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

プライマリのグリア細胞の in Vitro Neuroinflammation のモデリングのための三次元ハイドロゲル文化を改善

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

剛体の埋め込み固定し、ステンド グラス、全体、マイクロ コンピューター断層撮影によってセクション-無料の 3 D の組織学のためのミリ波スケール標本

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

長期ナノ材料暴露後のインビトロ遺伝子毒性試験のための高度な3D肝モデル

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...