マイクロウェルアレイは、米国エネルギー省基礎エネルギー科学局のナノフェーズ材料科学センターのユーザー施設部門のセンターで製作され、特性評価されました。この研究の資金援助は、オークリッジ国立研究所所長の研究開発基金を通じて提供された。著者らはまた、これらの研究で使用されたP.緑膿菌株の供給についてJ. Mougous Laboratory（Washington、Seattle、WA）に感謝したい 。

<ol>
	<li>Zhou, J., Deng, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochasticity,+succession,+and+environmental+perturbations+in+a+fluidic+ecosystem.">Stochasticity, succession, and environmental perturbations in a fluidic ecosystem.</a> Proc Natl Acad Sci. 111, E836-E845 (2014).</li><li>Valm, A. M., Welch, J. L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systems-level+analysis+of+microbial+community+organization+through+combinatorial+labeling+and+spectral+imaging.">Systems-level analysis of microbial community organization through combinatorial labeling and spectral imaging.</a> Proc Natl Acad Sci USA. 108 (10), 4152-4157 (2011).</li><li>Satoh, H., Miura, Y., Tsushima, I., Okabe, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Layered+structure+of+bacterial+and+archaeal+communities+and+their+in+situ+activities+in+anaerobic+granules.">Layered structure of bacterial and archaeal communities and their in situ activities in anaerobic granules.</a> Appl Environ Microbiol. 73 (22), 7300-7307 (2007).</li><li>Kim, H. J., Boedicker, J. Q., Choi, J. W., Ismagilov, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defined+spatial+structure+stabilizes+a+synthetic+multispecies+bacterial+community.">Defined spatial structure stabilizes a synthetic multispecies bacterial community.</a> Proc Natl Acad Sci USA. 105 (47), 18188-18193 (2008).</li><li>Nunan, N., Wu, K., Young, I. M., Crawford, J. W., Ritz, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+distribution+of+bacterial+communities+and+their+relationships+with+the+micro-architecture+of+soil.">Spatial distribution of bacterial communities and their relationships with the micro-architecture of soil.</a> FEMS Microbiol Ecol. 44, 203-215 (2003).</li><li>Grundmann, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+scales+of+soil+bacterial+diversity+-+The+size+of+a+clone.">Spatial scales of soil bacterial diversity - The size of a clone.</a> FEMS Microbiol Ecol. 48, 119-127 (2004).</li><li>Langenheder, S., Lindstrom, E. S., Tranvik, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+Function+of+Bacterial+Communities+Emerging+from+Different+Sources+under+Identical+Conditions.">Structure and Function of Bacterial Communities Emerging from Different Sources under Identical Conditions.</a> Appl Environ Microbiol. 72 (1), 212-220 (2006).</li><li>Camp, J. G., Kanther, M., Semova, I., Rawls, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+and+Scales+in+Gastrointestinal+Microbial+Ecology.">Patterns and Scales in Gastrointestinal Microbial Ecology.</a> Gastroenterology. 136 (6), 1989-2002 (2009).</li><li>Renner, L. D., Weibel, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physicochemical+regulation+of+biofilm+formation.">Physicochemical regulation of biofilm formation.</a> MRS Bull. 36 (5), 347-355 (2011).</li><li>Wessel, A. K., Hmelo, L., Parsek, M. R., Whiteley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Going+local:+technologies+for+exploring+bacterial+microenvironments.">Going local: technologies for exploring bacterial microenvironments.</a> Nat Rev Microbiol. 11 (5), 337-348 (2013).</li><li>Stacy, A., McNally, L., Darch, S. E., Brown, S. P., Whiteley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biogeography+of+polymicrobial+infection.">The biogeography of polymicrobial infection.</a> Nat Rev Microbiol. 14 (2), 93-105 (2015).</li><li>Hansen, R. R., Shubert, K. R., Morrell-Falvey, J. L., Lokitz, B. S., Doktycz, M. J., Retterer, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microstructured+block+copolymer+surfaces+for+control+of+microbe+adhesion+and+aggregation.">Microstructured block copolymer surfaces for control of microbe adhesion and aggregation.</a> Biosensors. 4 (1), 63-75 (2014).</li><li>Hansen, R. R., Hinestrosa, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lectin-functionalized+poly(glycidyl+methacrylate)-+block+-poly(vinyldimethyl+azlactone)+surface+scaffolds+for+high+avidity+microbial+capture.">Lectin-functionalized poly(glycidyl methacrylate)- block -poly(vinyldimethyl azlactone) surface scaffolds for high avidity microbial capture.</a> Biomacromolecules. 14 (10), 3742-3748 (2013).</li><li>Timm, C. M., Hansen, R. R., Doktycz, M. J., Retterer, S. T., Pelletier, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microstencils+to+generate+defined,+multi-species+patterns+of+bacteria.">Microstencils to generate defined, multi-species patterns of bacteria.</a> Biomicrofluidics. 9 (6), (2015).</li><li>Keymer, J. E., Galajda, P., Muldoon, C., Park, S., Austin, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+metapopulations+in+nanofabricated+landscapes.">Bacterial metapopulations in nanofabricated landscapes.</a> Proc Natl Acad Sci USA. 103 (46), 17290-17295 (2006).</li><li>Zhang, Q., Lambert, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acceleration+of+Emergence+of+Bacterial+Antibiotic+Resistance+in+Connected+Microenvironments.">Acceleration of Emergence of Bacterial Antibiotic Resistance in Connected Microenvironments.</a> Science. 333 (6050), 1764-1767 (2011).</li><li>Friedlander, R. S., Vlamakis, H., Kim, P., Khan, M., Kolter, R., Aizenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+flagella+explore+microscale+hummocks+and+hollows+to+increase+adhesion.">Bacterial flagella explore microscale hummocks and hollows to increase adhesion.</a> Proc Natl Acad Sci USA. 110 (14), 5624-5629 (2013).</li><li>Zhou, J., Liu, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+Assembly+Leads+to+Alternative+Communities+with+Distinct+Functions+in+a+Bioreactor+Microbial+Community.">Stochastic Assembly Leads to Alternative Communities with Distinct Functions in a Bioreactor Microbial Community.</a> MBio. 4 (2), 1-8 (2013).</li><li>van Vliet, S., Hol, F. J., Weenink, T., Galajda, P., Keymer, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+chemical+interactions+and+culture+history+on+the+colonization+of+structured+habitats+by+competing+bacterial+populations.">The effects of chemical interactions and culture history on the colonization of structured habitats by competing bacterial populations.</a> BMC Microbiol. 14 (1), 116 (2014).</li><li>Niepa, T. H. R., Hou, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+Nanoculture+as+an+Artificial+Microniche.">Microbial Nanoculture as an Artificial Microniche.</a> Sci Rep. 6, 30578 (2016).</li><li>Hansen, R. H., Timm, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+Assembly+of+Bacteria+in+Microwell+Arrays+Reveals+the+Importance+of+Confinement+in+Community+Development.">Stochastic Assembly of Bacteria in Microwell Arrays Reveals the Importance of Confinement in Community Development.</a> PLoS ONE. 11 (5), e0155080 (2016).</li><li>Hood, R. D., Singh, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Type+VI+Secretion+System+of+Pseudomonas+aeruginosa+Targets+a+Toxin+to+Bacteria.">A Type VI Secretion System of Pseudomonas aeruginosa Targets a Toxin to Bacteria.</a> Cell Host Microbe. 7 (1), 25-37 (2010).</li><li>LeRoux, M., Ja De Leon, ., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+single-cell+characterization+of+bacterial+interactions+reveals+type+VI+secretion+is+a+double-edged+sword.">Quantitative single-cell characterization of bacterial interactions reveals type VI secretion is a double-edged sword.</a> Proc Natl Acad Sci. 109 (48), 19804-19809 (2012).</li><li>Whitney, J. C., Beck, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+distinct+pathways+guide+effector+export+through+the+type+VI+secretion+system.">Genetically distinct pathways guide effector export through the type VI secretion system.</a> Mol Microbiol. 92 (3), 529-542 (2014).</li><li>Warrick, J. W., Timm, A., Swick, A., Yin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+for+Single-Cell+Kinetic+Analysis+of+Virus-Host+Interactions.">Tools for Single-Cell Kinetic Analysis of Virus-Host Interactions.</a> PLoS ONE. 11 (1), e0145081 (2016).</li><li>Zwietering, M. H., Jongenburger, I., Rombouts, F. M., Van't Riet, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+of+the+Bacterial+Growth+Curve.">Modeling of the Bacterial Growth Curve.</a> Appl Environ Microbiol. 56 (6), 1875-1881 (1990).</li><li>Halsted, M., Wilmoth, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+transparent+microwell+arrays+for+optical+monitoring+and+dissection+of+microbial+communities.">Development of transparent microwell arrays for optical monitoring and dissection of microbial communities.</a> J Vac Sci Technol B Nanotechnol Microelectron. 34 (6), 06KI03 (2016).</li></ol>

著者は何も開示することはない。

この記事では、マイクロウェルアレイデバイスと、細菌のコミュニティ開発のハイスループットと高含量の生細胞イメージングベースの解析を可能にするための実験プロトコルを紹介しました。ここでのデモの焦点は、接触仲介型VI型分泌が地域社会の発展に及ぼす影響を研究することであったが、広範囲の微生物群集と微生物 - 微生物相互作用の研究に柔軟に対応するように設計された。?...

微生物群は、環境の構造や細胞死、分裂、タンパク質濃度、オルガネラ数、突然変異1に関連する確率論的プロセスのような決定論的要因の両方によって形成される。自然環境の中で、これらの影響がコミュニティの構成と活動に与える個々の影響を解析することは、ほとんど不可能です。自然の構造によって隠され、化学的および生物学的環境に埋もれて、コミュニティのメンバーを特定し、自然環境内で時空間分布をさらに解決することは非常に困難です。それにもかかわらず、最近の努力は、地域の機能に関する空間的組織の重要性を強調し、進行中の研究2,3,4でメンバーの豊富さと組織の両方を考慮する必要性を指摘している。 それ（ 例えば、土壌構造、植物の根、海洋粒子、または腸の微絨毛）、酸素の存在または不存在、およびその存在が、病原性種はすべて、微生物群集の組成、構造、および機能に影響を及ぼす5,6,7,8,9,10,11。それにもかかわらず、これらの要因を捕らえることを怠った伝統的な文化の技法が引き続き普及している。コミュニティ構成（ 例えば、共依存種の存在）、物理的付着、シグナル伝達分子濃度、および直接的細胞 - 細胞接触は、微生物共同体を形成するためのすべての重要な因子であり、c予防的培養条件。これらの特性は、バルク液体培養または寒天プレート上で複製することは困難である。しかし、自然環境の主要な物理的および化学的特徴の複製を可能にするマイクロ流体、マイクロパターン形成、およびナノ製造技術の可能性は、多くの研究者が相互作用を研究する細菌群集を構築することを可能にした12,13,14。自然条件4,15,16,17,18,19,20を模倣する。 このプロトコールは、マイクロウェルアレイデバイスを作製する方法を記載し、そして、機能的にするために使用され得る詳細な実験手順を提供する単一ウェルのウェル内で細菌を増殖させることができます。この研究はまた、蛍光レポータータンパク質を産生するように改変された細菌が、時間の経過とともにウェル内の細菌増殖をモニターするためにどのように使用され得るかを実証する。同様のアレイを以前に提示し、 マイクロウェル中の緑膿菌（ P.緑膿菌）の単一種コロニーの成長を追跡することが可能であることを示した。ウェルサイズと播種密度を調整することにより、何千もの増殖実験の開始条件を並行して変化させることができ、初期接種条件がバクテリアの増殖能力にどのように影響するかを決定することができる21 。現在の研究では、複数のアレイの同時比較を可能にし、より堅牢な実験プロトコールを使用することにより、前の研究に基づいて構築されたわずかに改変されたマイクロウェルアレイのバージョンを使用する。この作品で使用されているアレイには、複数のサブアレイ、または配列ensem3つの異なるピッチ（ すなわち 、ウェル直径の2倍、3倍、および4倍）で配置された、直径15〜100μmの範囲の異なるサイズのウェルを含む。アレイはシリコン内にエッチングされ、シリコン注入アレイに封入された細菌の増殖は、培地注入アガロースゲルで被覆されたカバースリップでアレイを密封することによって可能になる。タイプ6分泌系を研究するために設計されたP.aeruginosa変異体がこのデモンストレーションで使用される。 ここで提示された結果は、マイクロウェルアレイ内のマルチメンバーコミュニティを分析し、化学環境の制御と探索を行っている間、研究者がバイオウムの豊富さと組織をインサイチュで監視できる究極の目標に向かっています。これは、最終的に、コミュニティの発展と継承を支配する「ルール」についての洞察を提供するはずです。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Parylene N</td>
			<td>Specialty Coating Systems</td>
			<td>CAS NO.:1633-22-3</td>
		</tr>
		<tr>
			<td>Parylene coater</td>
			<td>Specialty Coating Systems</td>
			<td>Labcoter 2 Parylene Deposition Unit PDS2010</td>
		</tr>
		<tr>
			<td>Silicon Wafer</td>
			<td>WRS Materials</td>
			<td>100mm diameter, 500-550&#956;m thickness, Prime, 10-20 resistivity, N/Phos&#60;100&#62;,</td>
		</tr>
		<tr>
			<td>adhesion promoter</td>
			<td>Shin-Etsu Microsci</td>
			<td>MicroPrime P20 adhesion promoter</td>
		</tr>
		<tr>
			<td>postive tone photoresist</td>
			<td>Rohm and Haas Electronics Materials LLC (Owned by Dow)</td>
			<td>Microposit S1818 Positive Photoresist (code 10018357)</td>
		</tr>
		<tr>
			<td>Quintel Contact Aligner</td>
			<td>Neutronix Quintel Corp</td>
			<td>NXQ 7500 Mask Aligner</td>
		</tr>
		<tr>
			<td>Reactive Ion Etching Tool</td>
			<td>Oxford Instruments</td>
			<td>Plasmalab System 100 Reactive Ion Etcher</td>
		</tr>
		<tr>
			<td>R2A Broth</td>
			<td>TEKnova</td>
			<td>R0005</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A9647</td>
		</tr>
		<tr>
			<td>Multimode Plate Reader</td>
			<td>Perkin Elmer</td>
			<td>Enspire, 2300-0000</td>
		</tr>
		<tr>
			<td>Fluorescent Microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti-U</td>
		</tr>
		<tr>
			<td>Automated Stage</td>
			<td>Prior</td>
			<td>ProScan III</td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>Nikon</td>
			<td>DS-QiMc</td>
		</tr>
		<tr>
			<td>Stage-top environmental control chamber</td>
			<td>In Vivo Scientific</td>
			<td>STEV ECU-HOC</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>ThermoFisher Scientific</td>
			<td>14190144</td>
		</tr>
		<tr>
			<td>UltraPure Agarose</td>
			<td>ThermoFisher Scientific</td>
			<td>16500500</td>
		</tr>
		<tr>
			<td>25 x 75 mm No. 1.5 coverslip</td>
			<td>Nexterion</td>
			<td>High performance #1.5H coverslips</td>
		</tr>
		<tr>
			<td>Fluorescence Reference Slides</td>
			<td>Ted Pella</td>
			<td>2273</td>
		</tr>
		<tr>
			<td>Physical Stylus Profilometer</td>
			<td>KLA Tencor</td>
			<td>P-6</td>
		</tr>
		<tr>
			<td>lab wipes</td>
			<td>Kimberly Clark</td>
			<td>Kimipe KIMTECH SCIENCE Brand, 34155</td>
		</tr>
		<tr>
			<td>commercial software</td>
			<td>Nikon</td>
			<td>NIS Elements</td>
		</tr>
		<tr>
			<td>Zeiss 710 Confocal Microscope</td>
			<td>Zeiss</td>
			<td></td>
		</tr>
		<tr>
			<td>filter cubes</td>
			<td>Nikon</td>
			<td>Nikon FITC (96311), Nikon Texas Red(96313)</td>
		</tr>
	</tbody>
</table>

assembly and tracking of microbial community development within a microwell array platform

微生物群集の発達は、コミュニティメンバーの空間分布および活動を劇的に変えることができる複雑な決定論的および確率論的因子の組み合わせに依存する。我々は、数千の細菌群を並行して迅速にアセンブルおよび追跡するために使用できるマイクロウェルアレイプラットフォームを開発しました。このプロトコルは、プラットフォームの有用性を強調し、プラットフォーム内の配列のアンサンブル内の単純な2メンバーのコミュニティの開発を光学的に監視するための使用方法を説明します。このデモでは、 緑膿菌の 2つの突然変異体を使用しています。これは、タイプVIの分泌病原性を研究するために開発された一連の突然変異体の一部です。 mCherryまたはGFP遺伝子のいずれかの染色体挿入物は、各マイクロウェル内のコミュニティメンバーの存在および位置をモニターするために使用され得る異なる発光波長を有する蛍光タンパク質の構成的発現を促進する。このプロトコルは、アレイのウェルに細菌の混合物を組み立て、時間経過に伴う各メンバー集団の相対的成長を測定するための経時的蛍光イメージングおよび定量的イメージ分析を使用する。マイクロウェルプラットフォームの播種およびアセンブリ、アレイ内の微生物群集の定量分析に必要なイメージング手順、およびすべて説明した微生物種間の相互作用を明らかにするために使用できる方法。

ここに提示された実験プラットフォームは、細菌群集のハイスループットおよびハイコンテント研究のために設計されています。このデザインにより、さまざまなサイズのウェルで成長する数千のコミュニティを同時に分析することができます。このマイクロウェルアレイ設計により、初期の播種密度、井戸の大きさ、および化学的環境への最終的なコミュニティ組?...

Watch this Scientific Journal Video about Assembly and Tracking of Microbial Community Development within a Microwell Array Platform at JoVE.com

マイクロウェル アレイ プラットフォーム内での微生物群集の発達の組み立てと追跡 |バイオエンジニアリング |ビデオ

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

微生物群集の発達は、環境構造、メンバーの豊富さ、形質および相互作用を含む複数の要因の組み合わせに依存する。このプロトコルは、ニッチサイズや閉じ込めなどの重要な要素を近似できるフェムトリットルウェルに含まれる数千のコミュニティを同時に追跡するための合成の微細加工環境を記述しています。

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 ...

assembly-tracking-microbial-community-development-within-microwell

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8.9K ビュー.  Oak Ridge National Laboratory. 微生物群集の発達は、環境構造、メンバーの豊富さ、形質および相互作用を含む複数の要因の組み合わせに依存する。このプロトコルは、ニッチサイズや閉じ込めなどの重要な要素を近似できるフェムトリットルウェルに含まれる数千のコミュニティを同時に追跡するための合成の微細加工環境を記述しています。 

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

Tracking Hypoxic Signaling within Encapsulated Cell Aggregates

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic &beta;-cells results in loss of insulin production and potentially lethal hyperglycemia. As an alternative treatment option to exogenous insulin injection, transplantation of functional pancreatic tissue has been explored1,2. This approach offers the promise of a more natural, long-term restoration of normoglycemia. Protection of the donor tissue from the host's immune system is required to prevent rejection and encapsulation is a method used to help achieve this aim.
Biologically-derived materials, such as alginate3 and agarose4, have been the traditional choice for capsule construction but may induce inflammation or fibrotic overgrowth5 which can impede nutrient and oxygen transport. Alternatively, synthetic poly(ethylene glycol) (PEG)-based hydrogels are non-degrading, easily functionalized, available at high purity, have controllable pore size, and are extremely biocompatible,6,7,8. As an additional benefit, PEG hydrogels may be formed rapidly in a simple photo-crosslinking reaction that does not require application of non-physiological temperatures6,7. Such a procedure is described here. In the crosslinking reaction, UV degradation of the photoinitiator, 1-[4-(2-Hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1-one (Irgacure 2959), produces free radicals which attack the vinyl carbon-carbon double bonds of dimethacrylated PEG (PEGDM) inducing crosslinking at the chain ends. Crosslinking can be achieved within 10 minutes. PEG hydrogels constructed in such a manner have been shown to favorably support cells7,9, and the low photoinitiator concentration and brief exposure to UV irradiation is not detrimental to viability and function of the encapsulated tissue10. While we methacrylate our PEG with the method described below, PEGDM can also be directly purchased from vendors such as Sigma.
An inherent consequence of encapsulation is isolation of the cells from a vascular network. Supply of nutrients, notably oxygen, is therefore reduced and limited by diffusion. This reduced oxygen availability may especially impact &beta;-cells whose insulin secretory function is highly dependent on oxygen11-13. Capsule composition and geometry will also impact diffusion rates and lengths for oxygen. Therefore, we also describe a technique for identifying hypoxic cells within our PEG capsules. Infection of the cells with a recombinant adenovirus allows for a fluorescent signal to be produced when intracellular hypoxia-inducible factor (HIF) pathways are activated14. As HIFs are the primary regulators of the transcriptional response to hypoxia, they represent an ideal target marker for detection of hypoxic signaling15. This approach allows for easy and rapid detection of hypoxic cells. Briefly, the adenovirus has the sequence for a red fluorescent protein (Ds Red DR from Clontech) under the control of a hypoxia-responsive element (HRE) trimer. Stabilization of HIF-1 by low oxygen conditions will drive transcription of the fluorescent protein (Figure 1). Additional details on the construction of this virus have been published previously15. The virus is stored in 10% glycerol at -80&deg; C as many 150 &mu;L aliquots in 1.5 mL centrifuge tubes at a concentration of 3.4 x 1010 pfu/mL. 
Previous studies in our lab have shown that MIN6 cells encapsulated as aggregates maintain their viability throughout 4 weeks of culture in 20% oxygen. MIN6 aggregates cultured at 2 or 1% oxygen showed both signs of necrotic cells (still about 85-90% viable) by staining with ethidium bromide as well as morphological changes relative to cells in 20% oxygen. The smooth spherical shape of the aggregates displayed at 20% was lost and aggregates appeared more like disorganized groups of cells. While the low oxygen stress does not cause a pronounced drop in viability, it is clearly impacting MIN6 aggregation and function as measured by glucose-stimulated insulin secretion15. Western blot analysis of encapsulated cells in 20% and 1% oxygen also showed a significant increase in HIF-1&alpha; for cells cultured in the low oxygen conditions which correlates with the expression of the DsRed DR protein.

A method for photo-encapsulation of cells in a crosslinked PEG hydrogel is described. Hypoxic signaling within encapsulated murine insulinoma (MIN6) aggregates is tracked using a fluorescent marker system. This system allows serial examination of cells within a hydrogel scaffold and correlation of hypoxic signaling with changes in cell phenotype.

カプセル化された細胞凝集体の中で低酸素シグナルの追跡

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic &beta;-cells results in loss of insulin production and potentially lethal ...

生物工学

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, each offering its own advantages and shortcomings5,6. Most prominently, due to the microscale device dimensions, a high modulus is required to facilitate implantation into living tissue. Conversely, the stiffness of the device should match the surrounding tissue to minimize induced local strain7-9. Therefore, we recently developed a new class of bio-inspired materials to meet these requirements by responding to environmental stimuli with a change in mechanical properties10-14. Specifically, our poly(vinyl acetate)-based nanocomposite (PVAc-NC) displays a reduction in stiffness when exposed to water and elevated temperatures (e.g. body temperature). Unfortunately, few methods exist to quantify the stiffness of materials in vivo15, and mechanical testing outside of the physiological environment often requires large samples inappropriate for implantation. Further, stimuli-responsive materials may quickly recover their initial stiffness after explantation. Therefore, we have developed a method by which the mechanical properties of implanted microsamples can be measured ex vivo, with simulated physiological conditions maintained using moisture and temperature control13,16,17. 
To this end, a custom microtensile tester was designed to accommodate microscale samples13,17 with widely-varying Young's moduli (range of 10 MPa to 5 GPa). As our interests are in the application of PVAc-NC as a biologically-adaptable neural probe substrate, a tool capable of mechanical characterization of samples at the microscale was necessary. This tool was adapted to provide humidity and temperature control, which minimized sample drying and cooling17. As a result, the mechanical characteristics of the explanted sample closely reflect those of the sample just prior to explantation.
The overall goal of this method is to quantitatively assess the in vivo mechanical properties, specifically the Young's modulus, of stimuli-responsive, mechanically-adaptive polymer-based materials. This is accomplished by first establishing the environmental conditions that will minimize a change in sample mechanical properties after explantation without contributing to a reduction in stiffness independent of that resulting from implantation. Samples are then prepared for implantation, handling, and testing (Figure 1A). Each sample is implanted into the cerebral cortex of rats, which is represented here as an explanted rat brain, for a specified duration (Figure 1B). At this point, the sample is explanted and immediately loaded into the microtensile tester, and then subjected to tensile testing (Figure 1C). Subsequent data analysis provides insight into the mechanical behavior of these innovative materials in the environment of the cerebral cortex.

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

A method is discussed by which the in vivo mechanical behavior of stimuli-responsive materials is monitored as a function of time. Samples are tested ex vivo using a microtensile tester with environmental controls to simulate the physiological environment. This work further promotes understanding the in vivo behavior of our material.

機械的に適応型ポリマーナノコンポジットの環境制御のMicrotensileテスト<em&gt; ex vivoで</em&gt;キャラ

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, ...

Synthesis of an Intein-mediated Artificial Protein Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 &#176;C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a &#34;docking station peptide&#34; into the hydrogel building block enables convenient incorporation of &#34;docking protein&#34;-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.

We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.

インテイン媒介人工タンパク質ヒドロゲルの合成

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

Development of an In Vitro Ocular Platform to Test Contact Lenses

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic physiological tear volumes.7 The traditional model also lacks the natural tear flow component and the blinking reflex, both of which are defining factors of the ocular environment. The development of a novel model is described in this study, which consists of a unique 2-piece design, eyeball and eyelid piece, capable of mimicking physiological tear volume. The models are created from 3-D printed molds (Polytetrafluoroethylene or Teflon molds), which can be used to generate eye models from various polymers, such as polydimethylsiloxane (PDMS) and agar. Further modifications to the eye pieces, such as the integration of an explanted human or animal cornea or human corneal construct, will permit for more complex in vitro ocular studies. A commercial microfluidic syringe pump is integrated with the platform to emulate physiological tear secretion. Air exposure and mechanical wear are achieved using two mechanical actuators, of which one moves the eyelid piece laterally, and the other moves the eyeballeyepiece circularly. The model has been used to evaluate CLs for drug delivery and deposition of tear components on CLs.

Development of an In Vitro Ocular Platform to Test Contact Lenses

Current in vitro models for evaluating contact lenses (CLs) and other eye-related applications are severely limited. The presented ocular platform simulates physiological tear flow, tear volume, air exposure and mechanical wear. This system is highly versatile and can be applied to various in vitro analyses with CLs.

開発<em&gt;インビトロ</em&gt;眼プラットフォームはコンタクトレンズをテストします

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

微細と3D微小血管の組立

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

粒子の空間追跡のための多重化された電子の検出とマイクロ流体プラットフォーム

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Rodent Behavioral Testing to Assess Functional Deficits Caused by Microelectrode Implantation in the Rat Motor Cortex

Medical devices implanted in the brain hold tremendous potential. As part of a Brain Machine Interface (BMI) system, intracortical microelectrodes demonstrate the ability to record action potentials from individual or small groups of neurons. Such recorded signals have successfully been used to allow patients to interface with or control&#160;computers, robotic limbs, and their own limbs. However, previous animal studies have shown that a microelectrode implantation in the brain not only damages the surrounding tissue but can also result in functional deficits. Here, we discuss a series of behavioral tests to quantify potential motor impairments following the implantation of intracortical microelectrodes into the motor cortex of a rat. The methods for open field grid, ladder crossing, and grip strength testing provide valuable information regarding the potential complications resulting from a microelectrode implantation. The results of the behavioral testing are correlated with endpoint histology, providing additional information on the pathological outcomes and impacts of this procedure on the adjacent tissue.

We have shown that a microelectrode implantation in the motor cortex of rats causes immediate and lasting motor deficits. The methods proposed herein outline a microelectrode implantation surgery and three rodent behavioral tasks to elucidate potential changes in the fine or gross motor function due to implantation-caused damage to the motor cortex.

ラット皮質運動野の微小電極注入による機能的障害を評価するためにテスト行動の齧歯動物

Medical devices implanted in the brain hold tremendous potential. As part of a Brain Machine Interface (BMI) system, intracortical microelectrodes ...

Prospecting Microbial Strains for Bioremediation and Probiotics Development for Metaorganism Research and Preservation

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. Globally, 4.5 billion people are economically dependent on the sea, where most of their livelihood is provided by coral reefs. Corals are of great importance and therefore their extinction leads to catastrophic consequences. There are several possible solutions to remediate marine pollutants and local contamination, including bioremediation. Bioremediation is the capacity of organisms to degrade contaminants. The approach presents several advantages, such as sustainability, relatively low cost, and the fact that it can be applied in different ecosystems, causing minimal impacts to the environment. As an extra advantage, the manipulation of endogenous microbiomes, including putative beneficial microorganisms for corals (pBMCs), may have probiotic effects for marine animals. In this context, the use of the two approaches, bioremediation and pBMC inoculation combined, could be promising. This strategy would promote the degradation of specific pollutants that can be harmful to corals and other metaorganisms while also increasing host resistance and resilience to deal with pollution and other threats. This method focuses on the selection of pBMCs to degrade two contaminants: the synthetic estrogen 17a-ethinylestradiol (EE2) and crude oil. Both have been reported to negatively impact marine animals, including corals, and humans. The protocol describes how to isolate and test bacteria capable of degrading the specific contaminants, followed by a description of how to detect some putative beneficial characteristics of these associated microbes to their coral host. The methodologies described here are relatively cheap, easy to perform, and highly adaptable. Almost any kind of soluble target compound can be used instead of EE2 and oil.

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. Bioremediation is the capacity of organisms to degrade contaminants. Here, we describe methodologies to isolate and test microbes presenting bioremediation ability and potential probiotic characteristics for corals.

メタ生物研究と保存のためのバイオレメディエーションおよびプロバイオティクス開発のための微生物株の探知

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. ...

Fabrication and Implementation of a Reference-Free Traction Force Microscopy Platform

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their microenvironment. While many approaches exist for measuring cell-induced material strain, here we provide a methodology for monitoring strain with sub-micron resolution in a reference-free manner. Using a two-photon activated photolithographic patterning process, we demonstrate how to generate mechanically and bio-actively tunable synthetic substrates containing embedded arrays of fluorescent fiducial markers to easily measure three-dimensional (3D) material deformation profiles in response to surface tractions. Using these substrates, cell tension profiles can be mapped using a single 3D image stack of a cell of interest. Our goal with this methodology is to make traction force microscopy a more accessible and easier to implement tool for researchers studying cellular mechanotransduction processes, especially newcomers to the field.

This protocol provides instructions for implementing multiphoton lithography to fabricate three-dimensional arrays of fluorescent fiducial markers embedded in poly(ethylene glycol)-based hydrogels for use as reference-free, traction force microscopy platforms. Using these instructions, measurement of 3D material strain and calculation of cellular tractions is simplified to promote high-throughput traction force measurements.

リファレンスフリートラクションフォース顕微鏡プラットフォームの製造と実装

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their ...

Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high encapsulation efficiency. Use of lipid nanoparticles (LNPs) to encapsulate nucleic acids is advantageous to protect the RNA or DNA from degradation, while also promoting cellular uptake. LNPs often contain multiple lipid components including an ionizable lipid, helper lipid, cholesterol, and polyethylene glycol (PEG) conjugated lipid. LNPs can readily encapsulate nucleic acids due to the ionizable lipid presence, which at low pH is cationic and allows for complexation with negatively charged RNA or DNA. Here LNPs are formed by encapsulating messenger RNA (mRNA) or plasmid DNA (pDNA) using rapid mixing of the lipid components in an organic phase and the nucleic acid component in an aqueous phase. This mixing is performed using a precise microfluidic mixing platform, allowing for nanoparticle self-assembly while maintaining laminar flow. The hydrodynamic size and polydispersity are measured using dynamic light scattering (DLS). The effective surface charge on the LNP is determined by measuring the zeta potential. The encapsulation efficiency is characterized using a fluorescent dye to quantify entrapped nucleic acid. Representative results demonstrate the reproducibility of this method and the influence that different formulation and process parameters have on the developed LNPs.

Lipid nanoparticles are developed using a microfluidic mixing platform approach for mRNA and DNA encapsulation.

マイクロ流体混合プラットフォームを用いた遺伝子導入のための脂質ナノ粒子の作製と特性化

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high ...