The authors would like to acknowledge the technical assistance of Alfred Gunasekaran in electron microscopy studies at the Institute of Micromanufacturing at Louisiana Tech University, and Dr. Jim McNamara for assistance with additional microscopy studies. The work described was supported in part by Louisiana board of Regents PKSFI Contract No. LEQSF (2007-12)-ENH-PKSFI-PRS-04 and the James E. Wyche III Endowed Professorship from Louisiana Tech University (to M.D.).

実験1.計画<ol><li>合成に必要な銅ナノ複合材料の量を決定します。その上、少量フラスコ（25 cm 2）で、または材料の調製において以下に示すように、より大きなフラスコの数を選択します。 </li><li>この合成には、5％のCO 2と少なくとも40％の湿度で37℃のインキュベーターを使用。このような培養器が利用可能であり、それは繰り返し合成（約24時間）の期間にわたって妨害されないことをことを確認してください。 注意：インキュベーターの繰り返し開閉は確かにナノコンポジット構造の変化した合成をもたらし得る温度変動の原因となります。 </li></ol>材料の作製<ol><li>合成が開始する直前に溶媒に固体材料を添加することにより、実験開始前に、新鮮なすべての材料を準備します。長時間Bの液体中にシスチンと銅の出発物質の原液を維持EFORE実験が推奨されていませんし、変数の結果につながる可能性があります。一度ベンダーから開く、パラフィルムで容器の上部をラップすることによって、乾燥出発材料保ちます。 注：以下のプロトコルは、シスチンの7μlの滅菌水の6643μL、およびCNPSの350μLを使用して25cm 2の細胞培養フラスコ内の反応のための例として使用されています。 </li><li> CNPSの少なくとも2ミリグラムを秤量することにより銅ナノ粒子の2 mg / mlの溶液を調製します。皮膚とのCNPSの可能性の接触を防ぐために、このステップの間に使い捨て手袋を着用してください。空の滅菌16ミリリットルのガラスバイアル中のナノ粒子を配置します。 <ol><li> CNPSを含むバイアルに、2 mg / mlの溶液を作製し、合成を開始する前に、ナノ粒子の分散を提供するために、20秒間、溶液をボルテックスするために、適切な音量に滅菌脱イオン水を加える（少なくとも1ミリリットルの合計容量を推奨します）。これはボルテックスで混合阻害するとして水をバイアルの半分以上の方法を記入しないでください。 CNPSウィルlはすぐにバイアルの底に沈殿し、（黒に灰色）色が暗い表示されます。 </li><li>合成の開始前にCNPSの最大分散を提供するために、室温で17分間、CNP液を超音波処理。定期的にCNPSは、超音波処理のために混合されていることを確認してください。成功した超音波処理後、CNPSを、少なくとも30分間、溶液中に懸濁したままで、溶液の色は暗くなります。 </li></ol></li><li>合成のための72.9 mg / mlの溶液を作製するためにシスチンの十分な質量を秤量します。シスチンは水に直接溶解しないので、帯電防止計量容器に秤量シスチンを配置します。 <ol><li>シスチンが完全に溶解するようにシスチンを含む計量容器に、滅菌、1MのNaOHの十分なボリュームを追加。たとえば、72.9 mg / mlの溶液を作製するために、1 M NaOHを100μlの完全シスチンの7.29ミリグラムを溶解します。 </li><li>無菌状態を維持するために、無菌のフロー組織培養フード中でこのステップを実行します。 注意：水酸化ナトリウム1でM濃度は苛性であるため、皮膚と濃NaOHとの接触を防ぐために、このステップの間に使い捨て手袋を着用します</li></ol></li><li>滅菌組織培養フードでの作業は、まず滅菌合成フラスコに滅菌水の6643μlのシスチンの7μlを添加し、フラスコのキャップで37℃のインキュベーター内で30分間インキュベートさせる効果的な提供するために、（ルーズ）通気混合。 CNPSは超音波処理工程の後に定住していますので、30秒間ボルテックスすることによって2 mg / mlのCNP液を再懸濁します。 <ol><li>合成を開始するために、25cm 2の細胞培養フラスコに滅菌水、50部CNPS、及び949の部品を1部品シスチンを組み合わせる次の成分比を維持するために（滅菌技術を使用して）の合成フラスコに十分なCNPの溶液を加えます。例えば、7ミリリットル合成ボリュームの、シスチン原液の7μL、CNPS350μlの、滅菌水の6643μLを兼ね備えています。それはSECURであるように、フラスコのキャップを取り付けて締めE。 </li><li>合成のためのすべてのコンポーネントを組み合わせた後、静かに4〜5回旋回することによりフラスコ中で混合します。 CO 2インキュベーター中でフラスコを置き、および合成の間、フラスコのうちのガス交換が存在することになるように、キャップを緩めて、フラスコをベント。 </li></ol></li><li>合成は約24時間インキュベーター内で実行することができます。合成の間、一つは、顕微鏡で眼によって、高度に線形複合体の形成を観察することができます。 注意：構造の形成のプロセスは、構造の検出が最初に困難であるという意味で突然発生する可能性があり、その外観は、高密度化に迅速に移行します。形成は、したがって、24時間前に発生することがあります。構造が大きくなると、その密度が増大したらプロセスは、目で観察することができます。構造体の生成は、後の時点で、顕微鏡下で目視により経時的に観察することができるが、連続的に合成条件を中断し、温度が低下につながります合成結果。 </li><li>しっかり合成フラスコをキャップし、冷蔵庫（4℃）で容器を保存することによって、バイオ複合材料の合成を終了します。一度生成構造は、少なくとも1年間はこの形で安定した状態を保ちます。利用コンポーネントを含む合成条件、合成の日付、および終了前に合成のインキュベーション時間でフラスコにラベルを付けます。 </li></ol>硫酸銅を使用した3合成<ol><li>硫酸銅塩とCNPSを置換することによって自己組織化合成を行います。無菌技術を使用して、2 mg / mlの溶液を作製するために滅菌脱イオン水の十分な量で硫酸銅の少なくとも2ミリグラムを溶かします。硫酸銅の結晶が容易にこの濃度で溶液中に行くが、必要に応じて、バイアルをボルテックスし、全ての結晶が溶解されることを保証するために目で点検します。 </li><li>前述のように硫酸銅を調製した後、合成を行うが、硫酸銅とCNPSを置き換えます。 注：出発物質として硫酸銅を用いて自己組織化ナノコンポジットはCNPSから合成した構造に比べて、最終的な形状が、はるかに一貫性のあることが見出されました。 </li><li> CNPコンポジット（ステップ2.6）の場合と同様に硫酸銅のバイオ複合材料の合成を終了し、4℃でそれらを長期的に保存します。 </li></ol> 4.特性とバイオ複合合成後の取り扱いについて<ol><li> CNPSから白色光顕微鏡9による硫酸銅からおよび電子顕微鏡9によって誘導されたバイオ複合材料を特徴づけます。 <ol><li>複合体は、フラスコが平坦敷設の数分以内に、フラスコの底面に沈降するように特性評価およびバイオ複合白色光顕微鏡による合成後の検査のために、倒立顕微鏡を使用し、次にフォーカスさせることができます。バイオ複合材料と液体媒体との間のコントラストを最大にするために顕微鏡で明視野設定を使用します。 CNPSと警官から派生複合硫酸ごとに両方の色が不透明に明確に表示されますが、未反応のCNPの凝集体は、色が非常に暗い表示されます。 <ol><li>複合材料の画像をキャプチャするために、顕微鏡に接続されたデジタルカメラを使用してください。個々の構造のための長さの範囲が観察されるであろう。 </li></ol></li><li>特性評価およびバイオ複合材料、合成後の検査のために、4℃で保存した後、フラスコを顕微鏡イメージングを行いながらフォーカス効果的な不明瞭ます冷蔵庫から取り出し、ときに最初に結露を形成するようにフラスコを少なくとも15分間、室温に来ることができます。 RTに平衡化させた後、顕微鏡の結像品質を最大にするために清潔なペーパータオルでフラスコの頂部および底部表面を拭きます。 </li><li>長期的に格納されている複合材料またはイメージング作業する場合、冷蔵庫にある間に形成複合材料の塊を解離するために30秒間、フラスコをボルテックス。ボルテックスした後、反転マイクを有する構造を検査roscope凝集体が解離していることを確認し、必要に応じてボルテックスを繰り返します。 </li><li> CNPSを使用して、指定された実験のために、合成の有効性を評価するために、反転白色光顕微鏡を使用してください。例えば、合成時のような異なるパラメータを有するフラスコからのCNP由来のバイオ複合材料に使用される合成フラスコ中の未反応CNPSの有無を記録します。 注：個々のCNPSを光学顕微鏡で観察するには小さすぎるが、未反応のCNPは、高アスペクト比、線形形式になります。合成に成功CNPコンポジットとは対照的に、ラウンド形状と​​暗いオブジェクトとして表示されます集約し、異なる長さの範囲を持っています。これは一旦形成された個々の構造に分散することが困難である高度に分岐した「ウニ」型構造、になりますように、終了までの時間の長すぎるために合成を行うことは避けてください。 </li><li>有効性を評価するために反転し、白色光顕微鏡を使用して硫酸銅を使用して、指定された実験の合成。硫酸銅は、このプロトコルを使用して溶液中に完全に移行しているため、溶液はCNPSを使用して、合成のソリューションよりも少ない暗い表示されます。そのような終了の前に、合成時と異なる合成条件でフラスコを比較することにより、硫酸銅複合体の大きさと範囲を文書。 注：正常に合成された複合材料は、異なる長さの範囲が表示されます。これは複合材料の高度に分岐した集合体になりますように、終了までの時間の長すぎるために合成を行う避け、「ウニ状」構造でなり、そのうちのいくつかと、一旦形成された個々の構造の中に分散させることは困難です。 </li></ol></li><li>バイオ複合材料を遠心管中の複合体の合成後、遠心分離ソリューションを集中させます。 15mlの遠心管にCNP由来の構造や硫酸銅由来の構造のいずれかの6ミリリットルを追加します。 10分間遠心室温で500×gでペレットを形成します。小さいボリュームの場合、0.6ミリリットルサイズのチューブに溶液中の構造物の500μlを添加します。少なくとも10分間、室温で2000×gで遠心分離し、ペレットを形成します。 <ol><li>十分な時間のために（microfugesのために少なくとも10分間）遠心分離した後、構造は慎重にペレット上記上澄み液を除去することにより濃縮して、チューブの底に観察可能なペレットを保存します。硫酸銅から派生バイオ構造は、（黒にグレー）暗いCNPSから派生した色および構造で青色に見えます。 </li><li>この管に、より複合材料を追加し、必要に応じて、構造物を濃縮するために、同じチューブ内で処理を繰り返します。濃縮されたペレットを分散させるために、10〜30秒のための所望のチューブに溶液の容量、および渦を追加します。 </li></ol></li><li>超音波処理の構造はかつて​​より低い値に構造体の平均集団サイズ（長さ）を移動し、形成されました。ルでのための滅菌脱イオン水や超音波処理中の場所の構造AST 10分。このプロセスを使用して、時間をかけて、構造は（テキストの図6を参照）断片化し、平均長さが小さくなります。反転白色光顕微鏡とデジタルカメラを使用して、さまざまな超音波処理時間を有する複合サイズのドキュメントの変更。 </li></ol>

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Authors have nothing to disclose.

CNPSを含むナノ材料の潜在的な毒性効果を評価するが、それは長期的に、CNPSが大きく、凝集形態に最初により分散微粒子分布（ 図2）から変換されたことが観察されました。いくつかの場合において、生物学的条件下で、細胞培養皿に製造されたこれらの高度に凝集形成は、「ウニ」を含む前述の銅を連想させる中央集合から非常に線状突起が形成された6。これは、ここ...

Copper is a highly reactive metal that in the biological world is essential in some enzyme functions 1,2, but in higher concentrations is potently toxic including in the nanoparticulate form 3,4. Concern over copper toxicity has become more relevant as CNPs and other copper-based nanomaterials are utilized, due to the increased surface area/mass for nanostructures. Thus, even a small mass of copper, in nanoparticle form, could cause local toxicity due to its ability to penetrate the cell and be broken down into reactive forms. Some biological species can complex with and chelate metal ions, and even incorporate them into biological structures as has been described in marine mussels 5. In studying the potential toxic effects of nanomaterials 4, it was discovered that over time, and under biological conditions used for typical cell culturing (37 &#176;C and 5% CO2), stable copper biocomposites could be formed with a high-aspect ratio (linear) structure.
By a process of elimination, the initial discovery of these linear biocomposites, which occurred in complete cell culture media, was simplified to a defined protocol of essential elements needed for the biocomposites to self-assemble. Self-assembly of two types of highly linear biocomposites was discovered to be possible with two starting metal components: 1) CNPs and 2) copper sulfate, with the common biological component being cystine. Although more complex, so called &#8220;urchin&#8221; and &#8220;nanoflower&#8221; type copper-containing structures with nanoscale and microscale features have been previously reported, these were produced under non-biological conditions, such as temperatures of 100 &#176;C or greater 6-8. To our knowledge, synthesis of individual, linear copper-containing nanostructures that are scalable in liquid phase under biological conditions has not been previously described. 
One of the starting materials utilized for synthesis of nanocomposites, namely CNPs, has been reported previously to be very toxic to cells 4. It has recently been reported that after the nanocomposites are formed, these structures are less toxic on a per mass basis than the starting NPs 9. Thus, the synthesis described here may be derived from a biological and biochemical reaction that has utility in stabilizing reactive copper species, both in the sense of transforming the NP form into larger structures and in producing composites less toxic to cells. 
In contrast to many other nanomaterial forms which are known to aggregate or clump upon interaction with biological liquid media 10,11, once formed, the highly linear composites described here avoid aggregation, possibly due to a redistribution of charge which has been previously reported 9. As detailed in the current work, this avoidance of aggregation is convenient for the purposes of working with the structures once formed for at least 3 reasons: 1) composite structures once formed may be concentrated using centrifugation and then easily dispersed again using vortex mixing; 2) formed structures can be decreased in average size by sonication for different periods of time; and 3) the formed linear structures may provide an additional tool for avoiding the recently described &#8220;coffee ring effect&#8221; 12 and thus provide a dopant for creating more evenly distributed coatings of materials, especially those containing spherical particulates.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Mini Vortexer</td>
			<td>VWR (https://us.vwr.com)</td>
			<td>58816-121</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator Model # 2425-2</td>
			<td>VWR (https://us.vwr.com)</td>
			<td>Contact vendor</td>
			<td>Current model calalog # 98000-360</td>
		</tr>
		<tr>
			<td>Eppendorf Centrifuge (Refrigerated Microcentrifuge)</td>
			<td>Labnet (http://labnetinternational.com/)</td>
			<td>C2500-R</td>
			<td>Model Prism R</td>
		</tr>
		<tr>
			<td>Cell Culture Centrifuge Model Z323K</td>
			<td>Labnet (http://labnetinternational.com/)</td>
			<td>Contact vendor</td>
			<td>Current model Z206A catalog # C0206-A</td>
		</tr>
		<tr>
			<td>Sonicator (Ultrasonic Cleaner)</td>
			<td>Branson Ultrasonics Corporation (http://www.bransonic.com/)</td>
			<td>1510R-MTH</td>
			<td></td>
		</tr>
		<tr>
			<td>Balance</td>
			<td>Sartorius (http://dataweigh.com)</td>
			<td></td>
			<td>Model CP225D similar model CPA225D</td>
		</tr>
		<tr>
			<td>Olympus IX51 Inverted Light Microscope</td>
			<td>Olympus (http://olympusamerica.com</td>
			<td>Contact vendor</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus DP71 microscope digital camera</td>
			<td>Olympus (http://olympusamerica.com</td>
			<td>Contact vendor</td>
			<td></td>
		</tr>
		<tr>
			<td>external power supply unit- white light for Olympus microscope</td>
			<td>Olympus (http://olympusamerica.com</td>
			<td>TH4-100</td>
			<td></td>
		</tr>
		<tr>
			<td>10x, 20, and 40x microscope objectives</td>
			<td>Olympus (http://olympusamerica.com</td>
			<td>Contact vendor</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning Electron Microscope</td>
			<td>Hitachi (http://hitachi-hitec.com/global/em/sem/sem_index.html)</td>
			<td>model S-4800</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission Electron Microscope</td>
			<td>Zeiss (http://zeiss.com/microscopy/en_de/products.html)</td>
			<td>model Libra 120</td>
			<td></td>
		</tr>
		<tr>
			<td>Table Top Work Station Unidirectional Flow Clean Bench</td>
			<td>Envirco (http://envirco-hvac.com)</td>
			<td>model PNG62675</td>
			<td>Used for sterile cell culture technique</td>
		</tr>
	</tbody>
</table>

generation of scalable, metallic high-aspect ratio nanocomposites in a biological liquid medium

このプロトコルの目的は、高アスペクト比の構造を有する2つの新規なバイオ複合材料の合成を記載することです。バイオ複合は、銅ナノ粒子（CNPS）または金属成分に寄与する硫酸銅のいずれかで、銅およびシスチンで構成されています。合成は、生物学的条件（37℃）で液体で行い、自己組織化複合体は、24時間後に形成されます。一旦形成されると、これらの複合材料は、両方の液体培地中で、乾燥した形で非常に安定しています。複合材料の長さは、数ミクロンから、直径25 nmの範囲をマイクロするナノスケールから。エネルギー分散型X線分光法（EDX）を用いて電界放出走査電子顕微鏡は、それが、最終的なナノ複合材料中の硫黄の供給源としてシスチンを確認し、出発CNP材料から不在であった硫黄は、NP由来の線状構造中に存在することを実証しました。これらの線状ナノ·マイクロ複合材料の合成、STRの長さの多様な範囲の間ucturesは、合成容器内で形成されています。合成後の液体混合物の超音波処理は、超音波処理時間の増加とともに平均の長さを減少させることにより、構造物の平均の大きさを制御するのを助けることが示されました。形成された構造は、非常に安定であり、凝集しない、液相中で形成されているので、遠心分離はまた、形成された複合物を濃縮し、分離を補助するために使用することができます。

図1は、この研究に記載の線状バイオ複合材料を形成するために、合成工程のフローチャート図を示しています。出発物質としてCNPSまたは硫酸銅を2 mg / mlの溶液を形成するために滅菌水と組み合わせて、この溶液を混合し、さらに混合物を提供するために超音波処理し、この銅溶液を合成するための以下の割合で混合される：949重量部、滅菌水：50部銅混合物：1部シスチン原液?...

Watch this Scientific Journal Video about Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium at JoVE.com

Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

ここでは、生物学的条件下で液体培地中で小説、高アスペクト比のバイオ複合材料を合成するためのプロトコルを提示します。バイオ複合材料は、それぞれ、直径および長さにマイクロメートルのナノメートルスケール。シスチンと組み合わせた銅ナノ粒子（CNPS）および硫酸銅は、合成のための重要なコンポーネントです。

生物学的液体培地中でスケーラブルな、メタリック高アスペクト比のナノコンポジットの生成

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper ...

generation-scalable-metallic-high-aspect-ratio-nanocomposites

Research

JoVE Journal

Bioengineering

9.0K ビュー.  Centenary College of Louisiana. ここでは、生物学的条件下で液体培地中で小説、高アスペクト比のバイオ複合材料を合成するためのプロトコルを提示します。バイオ複合材料は、それぞれ、直径および長さにマイクロメートルのナノメートルスケール。シスチンと組み合わせた銅ナノ粒子（CNPS）および硫酸銅は、合成のための重要なコンポーネントです。 

ビデオ: Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

ターゲット安定同位体希釈液体クロマトグラフィー - 質量分析のための細胞の脂質の抽出

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

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, ...

In situ TEM of Biological Assemblies in Liquid

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an additional application for these instruments- viewing viral assemblies in a liquid environment. This exciting and novel method of visualizing biological structures utilizes a recently developed microfluidic-based specimen holder. Our video article demonstrates how to assemble and use a microfluidic holder to image liquid specimens within a TEM. In particular, we use simian rotavirus double-layered particles (DLPs) as our model system. We also describe steps to coat the surface of the liquid chamber with affinity biofilms that tether DLPs to the viewing window. This permits us to image assemblies in a manner that is suitable for 3D structure determination. Thus, we present a first glimpse of subviral particles in a native liquid environment.

In situ TEM of Biological Assemblies in Liquid

Here we describe a procedure to image viral complexes in liquid at nanometer resolution using a transmission electron microscope.

その時 液体中の生物学的集合体のTEM

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an ...

Synthesis of Keratin-based Nanofiber for Biomedical Engineering

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of these porous nanofibers is of great interest due to their unique physiochemical properties. Here we elaborate on the fabrication of keratin containing poly (&#949;-caprolactone) (PCL) nanofibers (i.e., PCL/keratin composite fiber). Water soluble keratin was first extracted from human hair and mixed with PCL in different ratios. The blended solution of PCL/keratin was transformed into nanofibrous membranes using a laboratory designed electrospinning set up. Fiber morphology and mechanical properties of the obtained nanofiber were observed and measured using scanning electron microscopy and tensile tester. Furthermore, degradability and chemical properties of the nanofiber were studied by FTIR. SEM images showed uniform surface morphology for PCL/keratin fibers of different compositions. These PCL/keratin fibers also showed excellent mechanical properties such as Young&#39;s modulus and failure point. Fibroblast cells were able to attach and proliferate thus proving good cell viability. Based on the characteristics discussed above, we can strongly argue that the blended nanofibers of natural and synthetic polymers can represent an excellent development of composite materials that can be used for different biomedical applications.

Electrospun nanofibers have a high surface area to weight ratio, excellent mechanical integrity, and support cell growth and proliferation. These nanofibers have a wide range of biomedical applications. Here we fabricate keratin/ PCL nanofibers, using the electrospinning technique, and characterize the fibers for possible applications in tissue engineering.

医工学のためのケラチンベースのナノファイバーの合成

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of ...

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of smart soft devices for biomedical applications. Although a number of magnetically-responsive drug delivery systems have demonstrated efficacies through either in vitro proof of concept studies or in vivo preclinical applications, their use in clinical settings is still limited by their insufficient biocompatibility or biodegradability. Additionally, many of the existing platforms rely on sophisticated techniques for their fabrications. We recently demonstrated the fabrication of biodegradable, gelatin-based thermo-responsive microgel by physically entrapping poly(N-isopropylacrylamide-co-acrylamide) chains as a minor component within a three-dimensional gelatin network. In this study, we present a facile method to fabricate a biodegradable drug release platform that enables a magneto-thermally triggered drug release. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and thermo-responsive polymers within gelatin-based colloidal microgels, in conjunction with an alternating magnetic field application system.

We present a facile method to fabricate a biodegradable gelatin-based drug release platform that is magneto-thermally responsive. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and poly(N-isopropylacrylamide-co-acrylamide) within a spherical gelatin micro-network crosslinked by genipin, in conjunction with an alternating magnetic field application system.

制御された薬物放出のための磁場応答性ハイブリッドゼラチンマイクロゲルを交互に

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of ...

Preparation of Monodomain Liquid Crystal Elastomers and Liquid Crystal Elastomer Nanocomposites

LCEs are shape-responsive materials with fully reversible shape change and potential applications in medicine, tissue engineering, artificial muscles, and as soft robots. Here, we demonstrate the preparation of shape-responsive liquid crystal elastomers (LCEs) and LCE nanocomposites along with characterization of their shape-responsiveness, mechanical properties, and microstructure. Two types of LCEs &#8212; polysiloxane-based and epoxy-based &#8212; are synthesized, aligned, and characterized. Polysiloxane-based LCEs are prepared through two crosslinking steps, the second under an applied load, resulting in monodomain LCEs. Polysiloxane LCE nanocomposites are prepared through the addition of conductive carbon black nanoparticles, both throughout the bulk of the LCE and to the LCE surface. Epoxy-based LCEs are prepared through a reversible esterification reaction. Epoxy-based LCEs are aligned through the application of a uniaxial load at elevated (160 &#176;C) temperatures. Aligned LCEs and LCE nanocomposites are characterized with respect to reversible strain, mechanical stiffness, and liquid crystal ordering using a combination of imaging, two-dimensional X-ray diffraction measurements, differential scanning calorimetry, and dynamic mechanical analysis. LCEs and LCE nanocomposites can be stimulated with heat and/or electrical potential to controllably generate strains in cell culture media, and we demonstrate the application of LCEs as shape-responsive substrates for cell culture using a custom-made apparatus.

We demonstrate the preparation of siloxane-based and epoxy-based liquid crystal elastomers (LCEs) and LCE nanocomposites. The LCEs are characterized with respect to reversible strain, liquid crystal ordering, and stiffness. As a potential application, we demonstrate their use as shape-responsive substrates in a custom device for active cell culture.

モノドメイン液晶エラストマーおよび液晶エラストマーナノコンポジットの調製

LCEs are shape-responsive materials with fully reversible shape change and potential applications in medicine, tissue engineering, artificial muscles, and ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

モジュラー DNA デバイスの液体ハンドリング アセンブリを自動化

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

3D空間の細胞培養のための細胞骨格などの生体適合性液晶エラストマー発泡体の合成

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

癌細胞培養から採取した培地における液体クロマトグラフィー-質量分析法による選択キヌレニンの同時定量化

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

This protocol describes a dynamic culture system to produce controlled size aggregates of human pluripotent stem cells and further stimulate differentiation in cerebellar organoids under chemically-defined and feeder-free conditions using a single-use bioreactor.

ヒト多能性幹細胞からの成熟小脳オルガノイドのスケーラブルな生成と免疫染色による特徴付け

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...