本研究の一部は全米科学財団（NSF 1227359）、国立科学財団（EPS-1003907）、それぞれ国立科学財団（1007978）、およびWVU PSCoR、主催WVUのADVANCEオフィスによって資金を供給WVU EPSCoRプログラムによってサポートされていました。微細加工作業はWVU共用研究設備（クリーンルーム施設）とウェストバージニア大学のチップ研究所（マイクロチップラボ）でマイクロ流体統合セルラー研究で行われていた。共焦点イメージングは​​WVU顕微鏡イメージング施設で行われた。

1。フォトレジストマスターモールドのフォトリソグラフィ作製次のプロトコルは30-60ミクロンの直径とマイクロチャネルを作製するためのプロセスを示しています。小径（30μm未満）とマイクロチャネルを取得するには、フォトレジストのシングルスピンコーティングが必要とされている。 <ol><li>使用前に24時間クリーンルームに4℃で冷蔵庫からリフローフォトレジストを転送し、室温まで昇温することができます。 </li><li>シリコンウェハをきれいにし、それを脱水できるように150℃で1時間のためにそれを焼く。脱水は、シリコン基板上にフォトレジストの密着性を支援する。 </li><li>スピンコート次のレシピを使用してフォトレジストの第一層： </li></ol><table border="1"><tbody><tr><td> 手順 </td><td> 時間（秒） </td><td> 回転数（rpm） </td><td> 加速 </td></tr><tr><td> 1 </td><td> 2 &lt;/ TD&gt; <td> 300 </td><td>最高</td></tr><tr><td> 2 </td><td> 10 </td><td> 0 </td><td></td></tr><tr><td> 3 </td><td> 3 </td><td> 300 </td><td>最高</td></tr><tr><td> 4 </td><td> 60 </td><td> 600 </td><td>最高</td></tr><tr><td> 5 </td><td> 10 </td><td> 500 </td><td>最高</td></tr><tr><td> 6 </td><td> 10 </td><td> 600 </td><td>最高</td></tr></tbody></table><ol start="4"><li>その後、90秒110°Cの温度にホットプレート上でウェーハを焼く。ソフトベーク後のフォトレジストの厚さは20〜30程度になります。 </li><li>コー​​トの第一層に使用されるのと同じレシピに続く第2のフォトレジスト層をスピン。 </li><li>ソフトベーク90秒110°Cの温度にホットプレートの上に置くことによって、再びウェハ。このソフトベーク後のフォトレジストの厚さは40〜60程度になります。 </li><li> photor露光することによって正のマスターパターンを生成フィルムマスクを介し14,500 mJの個/ cm 2の露光量でUV光にesist。 </li><li>脱イオン（DI）水（1:2（v / v）の）で現像剤を希釈する。パターンが完全に開発されるまで、溶液中で繰り返しウエハを洗浄します。次いでDI水でウエハーを洗浄し、窒素ガスを用いて乾燥させる。 </li><li>リフロー：4分と溶媒の蒸発を防ぐために、ガラスのペトリ皿とカバーのため120°Cの温度にホットプレートの上に置き、ウェハを。ホットプレートからウェハを取り出し、それを室温まで冷却することができます。リフロー後のフォトレジストの厚さは50〜60程度になります。 </li></ol> 2。 PDMSマイクロチャネルネットワークのソフトリソグラフィーの作製<ol><li> 10:1の重量比（ベース：硬化剤）で、ポリジメチルシロキサン（PDMS）溶液を調製し、それを徹底的に自転·公転ミキサーを使って混ぜる。 </li><li>リフローされたフォトレジストマスターモールド上にPDMSソリューションをキャスト。ドガに15分間デシケーター中でキャストPDMSを置き。必要に応じて残りの気泡を除去するために窒素ガスを使用してください。 </li><li>それを治すために可能にするために3時間60°Cの温度でオーブンでPDMSを焼く。その後、マスターモールドから硬化PDMS層を除去。 </li><li>チャネルネットワークに穴をパンチすることにより入口/出口の穴を作成するために削っパンチャーを使用します。窒素ガスを用いたPDMSの表面を清掃してください。 </li><li> 4.5×10 -2トルの動作圧力でのプラズマクリーナーおよび3.5立方フィート /分の酸素流量内側に30秒間酸素プラズマを持つ2つのPDMS層を扱う。その後、光学顕微鏡下で手動でPDMSの表面を合わせます。アライメントのよりよい制御のために必要に応じて水を一滴も使用します。 </li><li>永久的な結合を実現するために30分間60℃のオーブンでデバイスを焼く。 </li></ol> 3。チップでHUVEC文化と種まき<ol><li>培養はL-グルタミンを有する培養培地でHUVECを、10％ウシ胎児血清（FBS）を補充した。目のために2〜5の電子実験通路を使用した。 </li><li> HUVECをが合流したら、37℃で2-6分間細胞をカウントし、最初のHEPESは（HEPES-BSS）緩衝生理食塩水で細胞を洗浄することによって、それらを収穫した後、トリプシン/ EDTAとインキュベートで細胞を扱うトリプシン処理が完了した後、トリプシン溶液で中和トリプシン/ EDTAを中和する。遠心分離し、それらを収集するために8％のデキストランを培養培地で細胞を懸濁する。デキストランは、良好な細胞播種や添付ファイルを助けるために媒体の粘度を増加させるために使用した。 </li><li> 5 4.5×10 -2トルの動作圧力とminと3.5の酸素流量フィート3 /分酸素プラズマでデバイスを扱う。その後、DI水でデバイスをロードし、殺菌のために層流バイオセーフティーフード内で8時間紫外線を扱う。 </li><li>細胞は準備ができて一日前に、1×リン酸塩を使用してデバイスを洗うフィブロネクチン（は100μg/ mlの、希でコート後、（1×PBS）緩衝生理食塩水1×PBSで貢献しました）、4℃で一晩冷蔵庫でインキュベート。 </li><li>フィブロネクチンコーティングした後、培養液でロードし1×PBSでデバイスを洗う。 15分間37℃の温度でデバイスをインキュベートする。 </li><li> 3-4×10 6細胞/ mlの細胞濃度を8％デキストラン培地でHUVEC細胞をロードする。デバイスの一つの入口で細胞20μlの液滴を置き、マイクロ流体チャネルに細胞を導入することを傾ける。 15-20分後、細胞は、チャネルの側壁に付着するために開始されます。デバイスの細胞のより均一な分布を作成するために15分毎に回転させます。必要に応じて、追加のローディングを行うことができる。 </li></ol> 4。長期灌流セットアップ静置培養の5-6時間後、添付されたHUVECを、完全に広げ始めます。 10μlの/時の定常流とリモコンシリンジポンプシステムを用いた灌流を設定します。灌流をfを調整することができる以上の流量、及び2週間〜4日の間の期間続くことができます。 5。細胞染色と顕微鏡の特性<ol><li>細胞はデバイス内部合流に到達すると、まず、徹底的にメディアを削除するには、1×PBSでデバイスを洗う。その後、希釈剤（4μL-1 ml）で希釈した赤色染料を使用してデバイスをロードします。セルローディング手順と同様の染料をロードします。室温で5分間、暗所で装置をインキュベートした後、染色を停止する培養液で装置を洗浄する。染料の長いインキュベーションは、細胞毒性とdisadhesionを引き起こす可能性があります。 </li><li> 1×PBS（1mL当たり2滴）で希釈し、青色色素をデバイスにロードします。室温で5分間、暗所でインキュベートし、その後完全に1×PBSで装置を洗浄する。 </li><li>倒立光学顕微鏡下で細胞染色を調べます。染色が良好であった場合は、（1×PBSで希釈した3.5％パラホルムアルデヒド）固定培地でマイクロチャネルをロード、その後水没培地と完全にアルミホイルでそれをカバーを固定でデバイス。 4℃の温度で冷蔵庫でデバイスを保管℃で乾燥し、退色からデバイスを防ぐことができます。固定デバイスは共焦点レーザ走査型顕微鏡によって行うことができる共焦点イメージングのための準備ができている。 </li></ol>

著者は、彼らが競合する経済的利益を持っていないことを宣言します。

<html> 1。マスターモールドの製造血管の形態計測のために設計し、指針の一つは、ネットワーク全体に血管径の分布は最小エネルギー配慮によって支配されていることを述べているマレーの法則16、として知られています。また、分岐で親血管の直径のキューブが娘血管の直径の立方体の和に等しいと述べている（ <img alt="式（1）" fo:content-width="0.9in" fo:src="/files/ftp_up...

循環系の一部として微小血管は、血液や組織の間の相互作用を媒介する、代謝活動を支援し、組織微小環境を定義し、多くの健康および病理学的条件において重要な役割を果たす。 in vitroでの機能的な微小血管の要約は、複雑な血管現象の研究のためのプラットフォームを提供することができます。しかしながら、このような内皮細胞の遊走アッセイ、内皮管形成アッセイ、およびラットおよびマウス大動脈輪アッセイなどのインビトロ微小血管アッセイ、従来の三次元（3D）ジオメトリおよび連続フロー制御に対してインビボ微小血管に再現することができない1-8。このような角膜血管新生アッセイ、ニワトリ絨毛尿膜血管新生アッセイ、およびマトリゲルプラグアッセイなどの動物モデルおよびin vivoアッセイを使用して、微小血管の研究は、多くの時間がかかり、コストが高く、観察や数量化に関して挑戦、および倫理的な問題1、9-13を上げる。 持っていないでしょうな、簡単かつ厳密に制御生物学的条件と動的流体環境などの研究14、、動物とし、 生体内での関連する高い実験的なコストと複雑さを節減しながらmicromanufacturingとマイクロ流体チップ技術の進歩は、生物医学科学の洞察の様々を有効にしている従来のマクロスケールの技術で可能であっ。 ここでは、 生体内微小血管内の3Dジオメトリを模倣し、フォトリソグラフィリフローフォトレジスト技術、ソフトリソグラフィー、マイクロフルイディクスとの組み合わせを使用して管理された連続的な灌流の流れの下で実行され内皮マイクロチャネルオンチップを構築するためのアプローチを提示する。

<table border="1">
	<tbody>
		<tr>
			<td>Reagent/Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reflow Photoresist</td>
			<td>AZ Electronic Materials</td>
			<td>AZP4620</td>
			<td></td>
		</tr>
		<tr>
			<td>Developer</td>
			<td>AZ Electronic Materials</td>
			<td>AZ 400K</td>
			<td></td>
		</tr>
		<tr>
			<td>PDMS</td>
			<td>Dow Corning Corporation</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr>
			<td>MCDB 131 Culture Medium</td>
			<td>Invitrogen</td>
			<td>10372-019</td>
			<td></td>
		</tr>
		<tr>
			<td>NacBlue Nuclei Staining</td>
			<td>Invitrogen</td>
			<td>H1399</td>
			<td></td>
		</tr>
		<tr>
			<td>PKH Red Stain</td>
			<td>Sigma</td>
			<td>MINI26 and PKH26GL</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Gibco</td>
			<td>PHE0023</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Sigma</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Invitrogen</td>
			<td>14040-133</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES Buffered Saline Solution</td>
			<td>Lonza</td>
			<td>CC-5024</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>Invitrogen</td>
			<td>25300-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin Neutralizing Solution</td>
			<td>Lonza</td>
			<td>CC-5002</td>
			<td></td>
		</tr>
		<tr>
			<td>PDMS Curing Agent</td>
			<td>Dow Corning Corporation</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary Human Umbilical Vein Endothelial Cells</td>
			<td>Lonza</td>
			<td>CC-2517</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Lonza</td>
			<td>14-501F</td>
			<td></td>
		</tr>
		<tr>
			<td>Diluent C</td>
			<td>Sigma</td>
			<td>CGLDIL</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst33342</td>
			<td>Invitrogen, Molecular Probes</td>
			<td>R37605</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran</td>
			<td>Sigma</td>
			<td>95771</td>
			<td></td>
		</tr>
		<tr>
			<td>3.5% Paraformaldehyde</td>
			<td>Electron Microscopy Science</td>
			<td>15710-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spinner</td>
			<td>Laurell Technologies Corporation</td>
			<td>WS-400BZ-6NPP/LITE</td>
			<td></td>
		</tr>
		<tr>
			<td>Desiccator</td>
			<td>BelArt Products</td>
			<td>999320237</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Pump System</td>
			<td>Harvard Apparatus</td>
			<td>PHD Ultra</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar Biosafety Hood</td>
			<td>Thermo Scientific</td>
			<td>1300 Series A2</td>
			<td></td>
		</tr>
		<tr>
			<td>Planetary Centrifugal Mixer</td>
			<td>Thinky</td>
			<td>ARE-310</td>
			<td></td>
		</tr>
		<tr>
			<td>Isotemp Oven</td>
			<td>Fisher Scientific</td>
			<td>13-246-516GAQ</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical Microscope</td>
			<td>Zeiss</td>
			<td>Invertoskop 40C</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma Cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-32G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hotplate</td>
			<td>Barnstead/Thermolyne Cimarec</td>
			<td>SP131635</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Scanning Confocal Microscope</td>
			<td>Zeiss</td>
			<td>LSM 510</td>
			<td></td>
		</tr>
	</tbody>
</table>

procedure for the development of multi-depth circular cross-sectional endothelialized microchannels-on-a-chip

インビボでの動物実験は、より時間がかかり、高価であり、観察および定量は非常に挑戦的であるための努力が微小血管の研究のためのin vitroアッセイの開発に焦点を当ててきた。 3次元（3D）ジオメトリに対してインビボ微小血管に表現し、連続的な流体の流れを提供する場合しかし、微小血管ビトロアッセイにおける従来は限界がある。フォトリソグラフィリフローフォトレジスト技術、ソフトリソグラフィー、マイクロフルイディクスとの組み合わせを使用して、我々は管理された連続灌流下で生体内微小血管および実行中の3Dジオメトリを模倣マルチ深円形の断面内皮マイクロチャネルオンチップを開発した流れ。ポジティブフォトレジストをリフロー半円形の断面マイクロチャンネルネットワークにマスターモールドを作製した。 replを2ポリジメチルシロキサン（PDMS）マイクロチャネルの整列及び接合によるマスター型からicated、円筒マイクロネットワークが作成されました。マイクロチャネルの直径は十分に制御することができる。また、チップ内部で播種し、一次ヒト臍帯静脈内皮細胞（HUVEC）は、細胞は4日〜2週間の間の時間期間の持続的な制御の下で灌流マイクロチャンネルの内面を内張りすることを示した。

マルチデプスマイクロチャネルネットワークを製造する当社のアプローチは、マイクロチャネルは、クロスセクション15を丸めていた、 生体内微小血管内の複雑な3D形状を模倣しています。さらに、チャネルと娘チャネルを分岐親の直径はおよそ全体のチャネル抵抗が低く、流動速度がネットワーク16-18全体より均一であるように要求されるレベルで流体の流れ?...

Watch this Scientific Journal Video about Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip at JoVE.com

Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip

マイクロチャネル·オン·チップ·プラットフォームは、フォトリソグラフィリフローフォトレジスト技術、ソフトリソグラフィー、マイクロフルイディクスとの組み合わせによって開発されました。内皮マイクロチャネルプラットフォームは、 インビボにおける微小血管の3次元（3D）ジオメトリを模倣する制御された連続かん流の流れの下で実行され、高品質かつリアルタイムイメージングを可能にし、微小血管の研究に適用することができる。

マルチ深円形の断面内皮マイクロチャンネル·オン·ア·チップの開発のための手順

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, ...

procedure-for-development-multi-depth-circular-cross-sectional

Research

JoVE Journal

Bioengineering

13.8K ビュー.  West Virginia University. マイクロチャネル·オン·チップ·プラットフォームは、フォトリソグラフィリフローフォトレジスト技術、ソフトリソグラフィー、マイクロフルイディクスとの組み合わせによって開発されました。内皮マイクロチャネルプラットフォームは、 インビボにおける微小血管の3次元（3D）ジオメトリを模倣する制御された連続かん流の流れの下で実行?...

ビデオ: Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip

Procedure for Lung Engineering

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths annually; chronic obstructive pulmonary disease is currently the fourth leading cause of death in the United States1. Contributing to this mortality is the fact that lungs do not generally repair or regenerate beyond the microscopic, cellular level. Therefore, lung tissue that is damaged by degeneration or infection, or lung tissue that is surgically resected is not functionally replaced in vivo. To explore whether lung tissue can be generated in vitro, we treated lungs from adult rats using a procedure that removes cellular components to produce an acellular lung extracellular matrix scaffold. This scaffold retains the hierarchical branching structures of airways and vasculature, as well as a largely intact basement membrane, which comprises collagen IV, laminin, and fibronectin. The scaffold is mounted in a bioreactor designed to mimic critical aspects of lung physiology, such as negative pressure ventilation and pulsatile vascular perfusion. By culturing pulmonary epithelium and vascular endothelium within the bioreactor-mounted scaffold, we are able to generate lung tissue that is phenotypically comparable to native lung tissue and that is able to participate in gas exchange for short time intervals (45-120 minutes). These results are encouraging, and suggest that repopulation of lung matrix is a viable strategy for lung regeneration. This possibility presents an opportunity not only to work toward increasing the supply of lung tissue for transplantation, but also to study respiratory cell and molecular biology in vitro for longer time periods and in a more accurate microenvironment than has previously been possible.

We have developed a decellularized lung extracellular matrix and novel biomimetic bioreactor that can be used to generate functional lung tissue. By seeding cells into the matrix and culturing in the bioreactor, we generate tissue that demonstrates effective gas exchange when transplanted in vivo for short periods of time.

肺のエンジニアリングのための手順

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths ...

生物工学

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

血液疾患における血管相互作用を研究するためのEndothelializedマイクロフルイディクス

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

Perfusion-based whole organ decellularization has recently gained interest in the field of tissue engineering as a means to create site-specific extracellular matrix scaffolds, while largely preserving the native architecture of the scaffold. To date, this approach has been utilized in a variety of organ systems, including the heart, lung, and liver 1-5. Previous decellularization methods for tissues without an easily accessible vascular network have relied upon prolonged exposure of tissue to solutions of detergents, acids, or enzymatic treatments as a means to remove the cellular and nuclear components from the surrounding extracellular environment6-8. However, the effectiveness of these methods hinged upon the ability of the solutions to permeate the tissue via diffusion. In contrast, perfusion of organs through the natural vascular system effectively reduced the diffusion distance and facilitated transport of decellularization agents into the tissue and cellular components out of the tissue. Herein, we describe a method to fully decellularize an intact porcine heart through coronary retrograde perfusion. The protocol yielded a fully decellularized cardiac extracellular matrix (c-ECM) scaffold with the three-dimensional structure of the heart intact. Our method used a series of enzymes, detergents, and acids coupled with hypertonic and hypotonic rinses to aid in the lysis and removal of cells. The protocol used a Trypsin solution to detach cells from the matrix followed by Triton X-100 and sodium deoxycholate solutions to aid in removal of cellular material. The described protocol also uses perfusion speeds of greater than 2 L/min for extended periods of time. The high flow rate, coupled with solution changes allowed transport of agents to the tissue without contamination of cellular debris and ensured effective rinsing of the tissue. The described method removed all nuclear material from native porcine cardiac tissue, creating a site-specific cardiac ECM scaffold that can be used for a variety of applications.

A method to rapidly and completely remove cellular components from an intact porcine heart through retrograde perfusion is described. This method yields a site specific cardiac extracellular matrix scaffold which has the potential for use in multiple clinical applications.

逆行性冠灌流によってブタ心臓の脱細胞化するための手順

Perfusion-based  whole organ decellularization has recently gained interest in the field of  tissue engineering as a means to create site-specific ...

Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is strongly regulated by the surrounding flow and nutritional environment. Biofilm growth also increases the heterogeneity of the local microenvironment by generating complex flow fields and solute transport patterns. To investigate the development of heterogeneity in biofilms and interactions between biofilms and their local micro-habitat, we grew mono-species biofilms of Pseudomonas aeruginosa and dual-species biofilms of P. aeruginosa and Escherichia coli under nutritional gradients in a microfluidic flow cell. We provide detailed protocols for creating nutrient gradients within the flow cell and for growing and visualizing biofilm development under these conditions. We also present protocols for a series of optical methods to quantify spatial patterns in biofilm structure, flow distributions over biofilms, and mass transport around and within biofilm colonies. These methods support comprehensive investigations of the co-development of biofilm and habitat heterogeneity.

Biofilms have complex interactions with their surrounding environment. To comprehensively investigate biofilm-environment interactions, we present here a series of methods to create heterogeneous chemical environment for biofilm development, to quantify local flow velocity, and to analyze mass transport in and around biofilm colonies.

バイオフィルムと生息地異質の共同開発を特徴付けるための方法

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

細胞内ターゲティングアプリケーションのための固体脂質ナノ粒子（SLNs）

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in complex mixtures. It is often used to analyze gasoline, jet fuel, diesel, bio-diesel and the organic fraction of bio-crude/bio-oil. In most of those analyses, the first dimension of separation is non-polar, followed by a polar separation. The aqueous fractions of bio-crude and other aqueous samples from biofuels production have been examined with similar column combinations. However, sample preparation techniques such as derivatization, solvent extraction, and solid-phase extraction were necessary prior to analysis. In this study, aqueous fractions obtained from the hydrothermal liquefaction of algae were characterized by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry without prior sample preparation techniques using a polar separation in the first dimension followed by a non-polar separation in the second. Two-dimensional plots from this analysis were compared with those obtained from the more traditional column configuration. Results from qualitative characterization of the aqueous fractions of algal bio-crude are discussed in detail. The advantages of using a polar separation followed by a non-polar separation for characterization of organics in aqueous samples by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry are highlighted.

A two-dimensional gas chromatography-time-of-flight mass spectrometry method is described for characterization of the aqueous fraction of bio-crude produced from hydrothermal liquefaction of algae. This protocol can also be employed to analyze the aqueous fraction of liquid products from fast pyrolysis, catalytic fast pyrolysis, catalytic deoxygenation and hydro-treating.

飛行時間型質量分析法と2Dガスクロマトグラフィーを用いて藻類の水熱液化からの水性画分の質的特性評価

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in ...

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., flasks or dishes). In fact, it is widely recognized that cells grown in flasks or dishes tend to de-differentiate and lose specialized features of the tissues from which they were derived. Currently, there are mainly two types of 3-D culture systems where the cells are seeded into scaffolds mimicking the native extracellular matrix (ECM): (a) static models and (b) models using bioreactors. The first breakthrough was the static 3-D models. 3-D models using bioreactors such as the rotating-wall-vessel (RWV) bioreactors are a more recent development. The original concept of the RWV bioreactors was developed at NASA&#39;s Johnson Space Center in the early 1990s and is believed to overcome the limitations of static models such as the development of hypoxic, necrotic cores. The RWV bioreactors might circumvent this problem by providing fluid dynamics that allow the efficient diffusion of nutrients and oxygen. These bioreactors consist of a rotator base that serves to support and rotate two different formats of culture vessels that differ by their aeration source type: (1) Slow Turning Lateral Vessels (STLVs) with a co-axial oxygenator in the center, or (2) High Aspect Ratio Vessels (HARVs) with oxygenation via a flat, silicone rubber gas transfer membrane. These vessels allow efficient gas transfer while avoiding bubble formation and consequent turbulence. These conditions result in laminar flow and minimal shear force that models reduced gravity (microgravity) inside the culture vessel. Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells and fibroblasts cultured under microgravity provided by the RWV bioreactor.

Cells growing in a three-dimensional (3-D) environment represent a marked improvement over cell cultivation in 2-D environments (e.g., flasks or dishes). Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa cultured under microgravity provided by rotating-wall-vessel (RWV) bioreactors.

微小重力下で増殖させたヒト腸粘膜の多細胞三次元器官型モデルの開発

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

マイクロウェルアレイプラットフォーム内での微生物コミュニティ開発の組み立てと追跡

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

Standard Test Method ASTM D 7998-19 for the Cohesive Strength Development of Wood Adhesives

The properties of cured wood adhesives are difficult to study because of the loss of water and other components to the wood, the influence of wood on the adhesive cure, and the effect of adhesive penetration on the wood interphase; thus, normal testing of a neat adhesive film is generally not useful. Most tests of wood adhesive bond strength are slow, laborious, can be strongly influenced by the wood and do not provide information on the kinetics of cure. Test method ASTM D 7998-19, however, can be used for fast evaluation of the strength of wood bonds. The use of a smooth, uniform, and strong wood surface, like maple face-veneer, and sufficient bonding pressure reduces the adhesion and wood strength effects on bond strength. This method has three main applications. The first is to provide consistent data on bond strength development. The second is to measure the dry and wet strengths of bonded lap shear samples. The third is to better understand the adhesive heat resistance by quickly evaluating thermal sensitivity and distinguishing between thermal softening and thermal degradation.

We present a procedure, ASTM D7998-19, for a rapid and more consistent evaluation of both dry and wet strength of adhesive bonds on wood. The method can also be used to provide information on strength development as a function of temperature and time or strength retention up to 250 &#176;C.

木材接着剤の凝集力開発のための標準試験方法ASTM D 7998-19

The properties of cured wood adhesives are difficult to study because of the loss of water and other components to the wood, the influence of wood on the ...

Asymmetrical Flow Field-Flow Fractionation for Sizing of Gold Nanoparticles in Suspension

Particle size is arguably the most important physico-chemical parameter associated with the notion of a nanoparticle. Precise knowledge of the size and size distribution of nanoparticles is of utmost importance for various applications. The size range is also important, as it defines the most &#8220;active&#8221; component of a nanoparticle dose.
Asymmetrical Flow Field-Flow Fractionation (AF4) is a powerful technique for sizing of particles in suspension in the size range of approximately 1&#8211;1000 nm. There are several ways to derive size information from an AF4 experiment. Besides coupling AF4 online with size-sensitive detectors based on the principles of Multi-Angle Light Scattering or Dynamic Light Scattering, there is also the possibility to correlate the size of a sample with its retention time using a well-established theoretical approach (FFF theory) or by comparing it with the retention times of well-defined particle size standards (external size calibration).
We here describe the development and in-house validation of a standard operating procedure (SOP) for sizing of an unknown gold nanoparticle sample by AF4 coupled with UV-vis detection using external size calibration with gold nanoparticle standards in the size range of 20&#8211;100 nm. This procedure provides a detailed description of the developed workflow including sample preparation, AF4 instrument setup and qualification, AF4 method development and fractionation of the unknown gold nanoparticle sample, as well as the correlation of the obtained results with the established external size calibration. The SOP described here was eventually successfully validated in the frame of an interlaboratory comparison study highlighting the excellent robustness and reliability of AF4 for sizing of nanoparticulate samples in suspension.

This protocol describes the use of Asymmetrical Flow Field-Flow Fractionation coupled with UV-vis detection for the determination of the size of an unknown gold nanoparticle sample.

懸濁液中の金ナノ粒子のサイズに対する非対称流量場流分画

Particle size is arguably the most important physico-chemical parameter associated with the notion of a nanoparticle. Precise knowledge of the size and ...