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1。プラズマ開始重合<ol><li>モノマー溶液の準備。 <ol><li> 200mlの水に分液ロートにヘキサン（100ml）で3回洗浄し、HEMA（0.718モル100ml）に溶解する。塩化ナトリウムで水相を飽和させ、ジエチルエーテル（50ml）でHEMAを抽出する。 MgSO 4で有機相を乾燥させ、 真空内で溶媒を除去し、（100ミリバール、40℃）。減圧下でHEMAを蒸留（15ミリバール、99℃）。 </li><li>第1.1.1項で生産阻害剤を含まないHEMAの0.62 Mのメタノール溶液を調製する。 1口フラスコに溶液30mlを注ぎ、1時間溶液を通してアルゴンをバブリングすることによって酸素を除去。 </li></ol></li><li>表面誘導重合。 <ol><li>位置プラズマチャンバ内に互いに隣接する2枚のポリカーボネート膜（ 図2）。気相の方を向いた膜の光沢面を配置します。 </li><li>ああにプラズマチャンバを接続5分間IGH真空（20ミリバール）。真空バルブを閉じ、アルゴンと酸素ガスに接続されており、15 SCCMのアルゴンと2.5 SCCM酸素と2時間、この混合物を用いて室内をパージされ、他のバルブを開きます。 </li><li>プラズマを開始し、（ポリカーボネートメンブレンの場合：12 W）の所望の電力への電力を削減し、プラズマで4分間の膜を処理する。対応するバルブを開くことにより、チャンバーとモノマー溶液を接続します。プラズマを切り、チャンバを排気。 </li><li>対応するバルブを開放することによってチャンバとモノマー溶液を接続し、チャンバー内に溶液を注ぐ。膜はモノマー溶液で覆われていることを確認してください。アルゴンに接続されたバルブを開いて、20℃（エアコンの部屋）で12時間、反応混合物を保存する。 </li><li>モノマー溶液を除去します。 5分間の超音波浴中でメタノールで膜を洗浄します。水で洗浄操作を繰り返します。 </li><li> メンブレンを乾燥さ2時間モレキュラーシーブ上で真空。 </li></ol></li></ol> 2。被覆膜の後置修飾<ol><li> tert-ブチル butylmetyletherおよびジメチルアミノピリジン（DMAP）（33、0.27ミリモル）、N、N-ジシクロヘキシルカルボジイミド（DCC）（0.27ミリモル55 mg）を、スピロベンゾピラン1の溶液（ 図1）（0.27ミリモル100mg）を調製（TBME）（12 ml）を加えた。 </li><li>丸底フラスコに保護スターラーバーと保護グリッドを配置します。フラスコを乾燥させ、アルゴンでフラスコをあふれさせる。 </li><li>コー​​ティングされた膜に続いてフラスコに溶液を注ぐ。 </li><li> 12時間室温で穏やかに撹拌する。 </li><li>溶液を除去し、5分間超音波浴中のtert-butylmetyletherを有する膜を洗う。エタノールと水で洗浄操作を繰り返します。 </li><li> 2時間モレキュラーシーブ上で真空中で膜を乾燥させます。 </li></ol> 3。表面張力の測定<ol><lI&gt;金属製のOリングの標準テープで膜を固定します。位置Oリングに接触していない膜表面の一部にナノ純水（3.3μl）を一滴を、。膜の5つの異なる箇所での接触角（CA）を測定します。 </li><li>サンプルの長期安定性を試験するために、3つの異なる0後の膜の斑点、1、2、3、7、14、および21日目に接触角を測定する。 </li></ol> 4。フォトクロミック膜の透過性試験<ol><li>水（12ml）でフランツ拡散セルのレセプターチャンバーを埋める。 </li><li>フランツ拡散セルに膜を固定します。膜受容体室の水と接触していることを確認してください。水溶液カフェイン溶液（; 3.0ミリリットル20 mM）を有するドナー室（膜の上室）埋める。白色光（ 図3）を有するドナーチャンバーの上部から、膜を照射する。受容体細胞からのサンプル（200μl）を収集し、FO200nmの細孔直径を有するr​​個のトラックエッジのポリカーボネート膜、試料を10分ごとに収集する。 </li><li>ステップ4.2で説明したように実験を繰り返す。しかし、全体の透過性試験の間にUV光（366ナノメートル、80 W / m 2）を用いて、膜を照射する。 </li><li>収集されたサンプルのカフェイン濃度の決定。 <ol><li> UV /可視分光計を用いて（0.05 mg / mlの1.5 mMの/ Lの間）に15種類のカフェイン濃度の較正曲線をプロットする。 293 nmで校正します。 </li><li>検量線を用いて収集したサンプルのそれぞれの濃度を測定。 </li><li> 対決定された濃度をプロットします。収集されたサンプルの時間。点を通る直線近似を行い、傾きからΔcのを決定します。 </li></ol></li></ol>

<ol>
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著者らは、開示することは何もありません。

プラズマ処理は、アルゴンイオン化によって引き起こされる紫色のガスを生成する。オレンジ色が漏れから望ましくない窒素の存在を示すであろう。プラズマ処理は、表面上にラジカルを形成するだけでなく、膜7,12をエッチングしないだけでなく。あまりにも多くのエッチングは、膜の透過性に影響を与えるであろう、かなりの細孔径を変更することができる。提示され、?...

材料のプラズマ改質は、多くの産業分野で重要なプロセスとなっています。材料のバルク特性を変えることなく、表面や表面 ​​の機能化の洗浄は、プラズマ処理表面科学1-8での本質的なプロセスになりました。 ポリマーのプラズマ処理はホモリシス結合開裂をもたらす。これは、ポリマー材料の縁にラジカルリッチ表面の形成をもたらす。酸素分子を含有するプラズマを用いて、表面を酸素に富むため、より親水性の9-11なる。しかし、表面の親水性は、時間12以上安定していない。長期安定性を高めるために、プラズマ処理された表面は、化学的に13-15後に、プラズマ処理中に変更することができる。この処理は、通常、プラズマ処理中のガス相への反応性モノマー種を添加することによって行われ、これらのモノマーは、その後重合さポリマー表面の作成ラジカルから。化学的処理は、不揮発性のモノマーを用いて実施されている場合、グラフトポリマーは、プラズマ改質後に行わなければならない。ラジカルが表面に形成された後、制御されたグラフト化を行うために、プラズマセットアップが制御された条件の下で、溶液中の12,16の表面からプラズマが開始した表面誘起重合を可能にする、記載されている。 プレゼンテーションでは、トラックエッジの高分子膜12,17の修正に焦点を当てています。これらの膜の表面張力を変更することによって、透過速度は、12を変化させることができる。これきれいで、高速プロセスは、高分子膜のバルク特性を変えることなく、膜表面全体を覆​​う非常に薄い層（&lt;5nm程度）の作成を可能にする。 、プラズマプロセス中のエッジングに、トラックエッジの膜の孔径がわずかに12を増大させる。エッジング率はdependiですポリマー上ngのとリニアな動作を持っています。 反応性官能基を有するモノマーを用いる場合、グラフトポリマーはさらに官能化することができる。これは、カルボン酸官能化スピロピランとPHEMA-グラフト膜の後置修飾によって実証される。スピロピランをUV光を照射するとメロシアニン種に変換することが知られているので、このことは、フォトクロミック表面をもたらす。スピロピラン形態は、可視光でメロシアニン形態（ 図1）18,19 を照射することによって再確立することができる。メロシアニン形態はスピロピラン状態よりも極性であるため、塗膜の表面張力は、光20をトリガすることができる。表面張力の変化は、水溶液に向かって膜の透過性に影響を与える。セットアップこれらの光応答性膜の透過性試験を行​​うためにどのように表示され、透過性抵抗の有意な変化は、（iを減少させる97％によりn透過性）が示される。このような膜は、薬物送達のセットアップでまたはスマート検知システムに組み込むことができる。 <img alt="図1" fo:content-width="5in" fo:src="/files/ftp_upload/51680/51680fig1highres.jpg" src="/files/ftp_upload/51680/51680fig1.jpg" /> スピロベンゾピラン化合物1の図1。光異性化。

<table border="0" cellpadding="0" cellspacing="0" style="width:943px;" width="943">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Name of Reagent/ Equipment</td>
			<td style="width:284px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">2-Hydroxyethyl methacrylate, 97%</td>
			<td style="width:284px;">Sigma-Aldrich</td>
			<td style="width:119px;">128635</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Hexane 99%</td>
			<td style="width:284px;">Biosolve</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Magnesium sulfate (MgSO4, anhydrous)</td>
			<td style="width:284px;">Sigma-Aldrich</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Methanol, 99%&#160;</td>
			<td style="width:284px;">Sigma-Aldrich</td>
			<td style="width:119px;">14262</td>
			<td>dried over molecular sieves</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">N,N-Dicylcohexylcarbodiimide, 99%</td>
			<td style="width:284px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Dimethyl aminopyridine, 99%</td>
			<td style="width:284px;">Sigma-Aldrich</td>
			<td style="width:119px;">107700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Tert-butylmethylether, 98%</td>
			<td style="width:284px;">Fluka</td>
			<td style="width:119px;">306975</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Polycarbonate membrane</td>
			<td style="width:284px;">Whatman</td>
			<td style="width:119px;"></td>
			<td>Nanopore Track Etched (TE) (1.0 &#956;m, 0.2 &#956;m, 0.1 &#956;m, 50 nm, 30 nm and 15 nm pore diameter; 47 mm or 25 mm membrane diameter)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Caffeine (reagent plus)</td>
			<td style="width:284px;">Sigma-Aldrich</td>
			<td style="width:119px;">C0750&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Franz diffusion cell (12 ml)</td>
			<td style="width:284px;">SES-Analysesysteme</td>
			<td style="width:119px;">6C010015</td>
			<td>15mm unjacheted Franz Cell, 12 ml Receptor volume, Flat ground, clear glass, stirbat and clamp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">UV-Lamp</td>
			<td style="width:284px;"></td>
			<td style="width:119px;"></td>
			<td>UV irradiation (366 nm, 15 W/m2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">White light lamp</td>
			<td style="width:284px;"></td>
			<td style="width:119px;"></td>
			<td>White light irradiation (500 W bulb)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">UV/Vis spectrophotometer</td>
			<td style="width:284px;">Varian 50Bio/50MPR</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:373px;">Polyester membranes</td>
			<td style="width:284px;">Sterlitech</td>
			<td style="width:119px;">PET0225100</td>
			<td>Polyester Membrane Filters, 0.2 &#956;m pore diameter, 25 mmm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Polyvinylidene fluoride membranes</td>
			<td style="width:284px;">Millipore</td>
			<td style="width:119px;"></td>
			<td>PVDF Membranes Durapore (0.22 &#956;m pore diameter; 47 mm membrane diameter)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Argon (99.9995%)</td>
			<td style="width:284px;">Alphagaz</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Dressler Cesar RF Power Generator</td>
			<td style="width:284px;"></td>
			<td style="width:119px;"></td>
			<td>Plasma chamber setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">MKS Multi Gas Controller 647C</td>
			<td style="width:284px;"></td>
			<td style="width:119px;"></td>
			<td>Plasma chamber setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">MKS Mass-Flow controllers</td>
			<td style="width:284px;"></td>
			<td style="width:119px;"></td>
			<td>Plasma chamber setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Vacuubrand RE 2.5 rotary vane vacuum pump</td>
			<td style="width:284px;"></td>
			<td style="width:119px;"></td>
			<td>Plasma chamber setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Contact angle measurement device Kr&#252;ss G10</td>
			<td style="width:284px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Balances Mettler Toledo AB204-S and Mettler ME30</td>
			<td style="width:284px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of light-responsive membranes by a combined surface grafting and postmodification process

市販のトラックエッジのポリマー膜の表面張力を修正するために、表面開始重合の方法が提示される。膜表面からの重合は、2 - ヒドロキシエチルメタクリレート（HEMA）のメタノール溶液で膜表面を反応させ、続いて、膜のプラズマ処理によって誘導される。特別な注意が前に表面上に重合プラズマ処理のためのプロセスパラメータに与えられる。例えば、膜の異なるタイプ（ 例えば、ポリエステル、ポリカーボネート、ポリフッ化ビニリデン）に対するプラズマ処理の影響が検討されている。さらに、表面グラフト化膜の経時安定性は、接触角測定によって示されている。このように、ポリ（2 - ヒドロキシエチルメタクリレート）（PHEMA）をグラフトすると、表面はさらに、所望の物質のカルボン酸官能基を有するポリマーのアルコール部分エステル化することにより修飾することができる。これらの反応は、したがって、膜表面の官能化のために使用することができる。例えば、膜の表面張力を変化させることができる、または提示光反応性等の所望の機能を挿入することができる。これは、光応答性膜をもたらすスピロベンゾピラン部官能化カルボン酸とPHEMAを反応させることによって実証される。溶媒の選択は、後置修飾工程において主要な役割を果たしており、本稿で詳しく説明されている。そのような官能化された膜の透過測定は外部光源にフランツセルを用いて行われる。 UV-可視範囲の光の波長を変化させることにより、水性カフェイン溶液の透磁率の変化が観察される。

エッチング速度は、異なる期間の後に、膜を秤量することによって追跡することができる。 図4から分かるように、エッチング速度は、ポリエステル、ポリフッ化ビニリデン、ポリカーボネートのために、以下の質量損失対エッチング時間の線形相関の傾きから求めることができるリニアエッチング速度を、膜。 図4に示すように、ポリカーボネート膜は、す?...

Watch this Scientific Journal Video about Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process at JoVE.com

Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process

プラズマ誘導重合手順は、高分子膜の表面開始重合について記載されている。フォトクロミック物質とグラフトポリマーの更後置修飾は、光応答性膜の透過性測定を行うプロトコルが提示される。

複合表面グラフトと後置修飾法による光応答膜の調製

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is ...

preparation-light-responsive-membranes-combined-surface-grafting

Research

JoVE Journal

Bioengineering

11.7K ビュー.  Empa, Swiss Federal Laboratories for Materials Science and Technology. プラズマ誘導重合手順は、高分子膜の表面開始重合について記載されている。フォトクロミック物質とグラフトポリマーの更後置修飾は、光応答性膜の透過性測定を行うプロトコルが提示される。 

ビデオ: Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process

Measurement of Aggregate Cohesion by Tissue Surface Tensiometry

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium. We have developed tissue surface tensiometry (TST) specifically to measure the surface free energy of interaction between cells. The biophysical concepts underlying TST have been previously described in detail1,2. The method is based on the observation that mutually cohesive cells, if maintained in shaking culture, will spontaneously assemble into clusters. Over time, these clusters will round up to form spheres. This rounding-up behavior mimics the behavior characteristic of liquid systems. Intercellular binding energy is measured by compressing spherical aggregates between parallel plates in a custom-designed tissue surface tensiometer. The same mathematical equation used to measure the surface tension of a liquid droplet is used to measure surface tension of 3D tissue-like spherical aggregates. The cellular equivalent of liquid surface tension is intercellular binding energy, or more generally, tissue cohesivity. Previous studies from our laboratory have shown that tissue surface tension (1) predicts how two groups of embryonic cells will interact with one another1-5, (2) can strongly influence the ability of tissues to interact with biomaterials6, (3) can be altered not only through direct manipulation of cadherin-based intercellular cohesion7, but also by manipulation of key ECM molecules such as FN8-11 and 4) correlates with invasive potential of lung cancer12, fibrosarcoma13, brain tumor14 and prostate tumor cell lines15. In this article we will describe the apparatus, detail the steps required to generate spheroids, to load the spheroids into the tensiometer chamber, to initiate aggregate compression, and to analyze and validate the tissue surface tension measurements generated.

We describe a method of measuring binding energy, expressible as tissue surface tension, between cells within 3D tissue-like aggregates. Differences in tissue surface tension have been demonstrated to correlate with invasiveness of lung, muscle, and brain tumors, and are fundamental determinants of establishing spatial relationships between different cell types.

組織の表面張力測定ごとの集計凝集力の測定

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium.  We ...

生物工学

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

3次元細胞培養のための自己申告足場

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

マウスモデルにおける組織工学血管の生成とグラフト

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

三層のための3つのユニークな環境を提供するために、エレクトロスピニング法を適応させます<em&gt;インビトロ</em&gt;気道壁のモデル

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

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 ...

Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using an automated robotic dispensing system. The particular bioink was selected as it allows cells to sediment towards and sense the features. The dispensing system bioprints viable cells in hydrogel bioinks using a backpressure assisted print head. However, by replacing the print head with a sharpened stylus or scalpel, the dispensing system can also be employed to create topographical cues through surface etching. The stylus movement can be programmed in steps of 10 &#181;m in the X, Y and Z directions. The patterned grooves were able to orientate mesenchymal stem cells, influencing them to adopt an elongated morphology in alignment with the grooves&#39; direction. The patterning could be designed using plotting software in straight lines, concentric circles, and sinusoidal waves. In a subsequent procedure, fibroblasts and mesenchymal stem cells were suspended in a 2% gelatin bioink, for bioprinting in a backpressure driven extrusion printhead. The cell bearing bioink was then printed using the same programmed coordinates used for the etching. The bioprinted cells were able to sense and react to the etched features as demonstrated by their elongated orientation along the direction of the etched grooves.

This protocol describes a bioprinting methodology using an automated robotic depositing system that incorporates etched topographical guidance cues with the precision deposition of a cell bearing hydrogel bioink. The printed cells are directly delivered to the etched features and are able to sense and orientate with them.

細胞の表面のガイダンスとBioprintingための自動ロボット調剤技術

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

水質浄化のための膜のような防汚特性を持つハイドロゲルの合成

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

ナノ製剤中のエンドトキシン検出カブトガニ リムラス ライセート (ラル) 試金を使用して

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Assembly and Characterization of Biomolecular Memristors Consisting of Ion Channel-doped Lipid Membranes

The ability to recreate synaptic functionalities in synthetic circuit elements is essential for neuromorphic computing systems that seek to emulate the cognitive powers of the brain with comparable efficiency and density. To date, silicon-based three-terminal transistors and two-terminal memristors have been widely used in neuromorphic circuits, in large part due to their ability to co-locate information processing and memory. Yet these devices cannot achieve the interconnectivity and complexity of the brain because they are power-hungry, fail to mimic key synaptic functionalities, and suffer from high noise and high switching voltages. To overcome these limitations, we have developed and characterized a biomolecular memristor that mimics the composition, structure, and switching characteristics of biological synapses. Here, we describe the process of assembling and characterizing biomolecular memristors consisting of a 5 nm-thick lipid bilayer formed between lipid-functionalized water droplets in oil and doped with voltage-activated alamethicin peptides. While similar assembly protocols have been used to investigate biophysical properties of droplet-supported lipid membranes and membrane-bound ion channels, this article focuses on key modifications of the droplet interface bilayer method essential for achieving consistent memristor performance. Specifically, we describe the liposome preparation process and the incorporation of alamethicin peptides in lipid bilayer membranes, and the appropriate concentrations of each constituent as well as their impact on the overall response of the memristors. We also detail the characterization process of biomolecular memristors, including measurement and analysis of memristive current-voltage relationships obtained via cyclic voltammetry, as well as short-term plasticity and learning in response to step-wise voltage pulse trains.

Soft, low-power, biomolecular memristors leverage similar composition, structure, and switching mechanisms of bio-synapses. Presented here is a protocol to assemble and characterize biomolecular memristors obtained from insulating lipid bilayers formed between water droplets in oil. The incorporation of voltage-activated alamethicin peptides results in memristive ionic conductance across the membrane.

アセンブリおよびイオン チャネルをドープした脂質膜から成る分子 Memristors のキャラクタリゼーション

The ability to recreate synaptic functionalities in synthetic circuit elements is essential for neuromorphic computing systems that seek to emulate the ...

Growth of Human and Sheep Corneal Endothelial Cell Layers on Biomaterial Membranes

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is deleterious for the cells as it reduces their ability to form a mature and functional layer. Here, we present a method for establishing and subculturing human and sheep corneal endothelial cell cultures that minimizes the loss of cell-to-cell contact. Explants of corneal endothelium/Descemet&#39;s membrane are taken from donor corneas and placed into tissue culture under conditions that allow the cells to collectively migrate onto the culture surface. Once a culture has been established, the explants are transferred to fresh plates to initiate new cultures. Dispase II is used to gently lift clumps of cells off tissue culture plates for subculturing. Corneal endothelial cell cultures that have been established using this protocol are suitable for transferring to biomaterial membranes to produce tissue-engineered cell layers for transplantation in animal trials. A custom-made device for supporting biomaterial membranes during tissue culture is described and an example of a tissue-engineered graft composed of a layer of corneal endothelial cells and a layer of corneal stromal cells on either side of a collagen type I membrane is presented.

This protocol describes the critical steps required to establish and grow corneal endothelial cell cultures from explants of human or sheep tissue. A method for subculturing corneal endothelial cells on membranous biomaterials is also presented.