This study was supported by a Singapore National Research Foundation (NRF) Grant NRF2012NRF-POC001-043.

1.フォトマスク製造<ol><li>石英槽に浸漬し、120℃で20分間：ピラニア溶液（1比2でH 2 SO 4 / H 2 O 2）を用いて4 &quot;のガラスウェハをきれい。 </li><li>電子ビーム蒸発器7（ 図1A）を使用して、ガラスウェハ上にクロム/金（AuのCR /1μmの30 nm）の層の層を堆積させます。 <ol><li>電子ビーム蒸発器でウェーハを配置します。真空が5×10 -6トルに達すると、高電圧源（10キロボルト）をオンにしてください。モニターコントロールパネルにより厚さを制御します。 </li><li>電子ビーム銃を用いて30秒（ウエハ上への堆積を回避するために）シャッター「OFF」に維持するため、各材料プリクリーン。 </li></ol></li><li>ガラスの深いウェットエッチング用のCr / Auのフォトレジストマスキング層を生成します。 <ol><li>スピンスプレーコーターシステムを用いて3000rpmで30秒間溶液5mlを回転させることにより厚さ2μmのフォトレジストを適用します。1.5分間、100℃のホットプレート上にフォトレジストをベーキング。 </li><li>公開とハードホットプレート上で30分間120℃でフォトレジストを焼きます。これは、疎水性表面と金属層にフォトレジストの強い接着力を発生させることが重要です。パターンフォトレジストマスク8,9を介してCr及びAuのエッチング液を使用してのCr / Au層。 </li></ol></li><li>非パターン化ガラス表面の保護のため、一時的にダミーシリコンウエハ、ガラスウエハを接合。9 <ol><li> 110℃のホットプレート上でガラスウエハを配置し、（ウェハの全表面をワックスで被覆されているように）ガラスウエハの反対側に、ワックスを溶融します。 </li><li>ガラスウェハに接触してダミーのシリコンウェハを置き、余分なワックスを除去するために押します。ワックスのこぼれを回避するために、ホットプレート上でクリーンルームティッシュペーパーを置きます。 </li></ol></li><li>最適化されたフッ酸を用いたレンズの等方性エッチングを行う（49％v / v）の8.5磁気スターラー分のHCl 10プレゼンス生成レンズの良好な表面品質を達成するために重要である：（1〜10の体積比で）と塩酸（37％v / v）の溶液。 <ol><li>エッチングレートが7ミクロン/分であることを確認してください。エッチング液を200mlの全容量を使用します。プラスチック容器にエッチングを行い、この処理ステップのための安全対策を取ります。 </li><li> RTですすぎ、さらに乾燥することにより、脱イオン（DI）水でウェハを清掃してください。 </li></ol></li><li>処理完了後、ダミーのシリコンウェハからガラスウェハを分離し、15秒間100℃のホットプレートを用いてワックスを温めます。ワックスはこの温度で​​溶融するように、ダミーのシリコンウェハからガラスウェハを取り外します。 </li><li>超音波タンク内の80℃の溶媒としてのNメチル-2-ピロリドンを用いて、1時間超音波を使用して、レンズの端に残っているワックス、フォトレジストオーバーハングのCr / Au層を削除します。</li><li>フォトマスク11上に製造されたマイクロレンズのPDMS型レプリカを作成します。 </li><li>フォトマスクの寸法（長さおよび幅）を特徴づけるとマイクロレンズPDMS型（深さと直径）は、それぞれ、走査型電子顕微鏡および実体顕微鏡を用いてレプリカ。12-14 </li></ol> 2. MNのシャフト製作<ol><li>ガラスの両側に取り付けられたガラススライドを使用して0.9センチメートル×2.5センチメートルの空洞を作成します。いずれかの側に積層ガラススライドの数は、スペーサの厚さ（ 図1B）と呼ばれるキャビティの高さを決定します。 </li><li> 0.5％w / wの2-ヒドロキシ-2-メチル - プロピオフェノン（HMP）を有するポリ含有プレポリマー溶液（エチレングリコール）ジアクリレート（PEGDA、MW = 258 Da）での薄層を適用することにより、スライドガラスの各層を確保します2秒間高強度の紫外線を設定（UV）光を照射することにより、その後、スライドガラス。 </li><li> photomaを配置SK空洞の内部に面するのCr / Auをコーティングした表面に（以前に製造されました）。空洞壁の側面は、フォトマスクに埋め込まれたレンズを曖昧にされていないことを確認してください。 </li><li> Cr / Auをコーティングした表面は、目に見える気泡ない溶液と接触するまでプレポリマー溶液を用いて空洞を満たします。 </li><li> 320から500 nmでのUVフィルターの範囲を有するUV硬化ステーションを使用して、UV光源から3.5センチ間隔で1秒間、所望の強度の高強度のUV光を照射してセットアップ。 UV光プローブでコリメートアダプターを使用してください。 </li><li>放射計を用いて使用されるUV光の強度を測定します。 </li><li>紫外線露光後、MNのの配列を使用してフォトマスクを削除します。再利用のために、その元の容器に戻し工程で重合されていない過剰プレポリマー溶液を注ぎます。 </li><li>製造業者の指示に従って実体顕微鏡を用いてのMNの長さおよび先端の直径を定量化します。 </li></ol><pクラス= &quot;jove_title&quot;&gt; 3。 MNバッキング層の作製<ol><li> 図1Cに示すように鉗子のMNを配置して（以前に製造された）24ウェルプレートのウェルに、フォトマスク上に取り付けられています。 </li><li>針が所望の高さに水没するまで十分にプレポリマー溶液の - （400μL300）指定されたボリュームを追加します。この量は、得られたバッキング層の厚さを決定します。 </li><li> 10.5センチメートル離れて1秒の持続時間のための紫外線源から、高強度のUV光（15.1 W / cm 2）としてセットアップを照射。 </li><li>鋭い刃を使用してフォトマスクからMNアレイ上にバッキング層を分離します。 </li><li>製造業者の指示に従って実体顕微鏡を用いて、バッキング層とのMNの長さ、先端の直径とベース直径を定量化します。 </li></ol>

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The authors declare no conflict of Interest.

MNのアレイの製造について上記したプロトコルは、約1 cm 2のMNのアレイを製造するために提示されています。アレイは、大きなサイズの空洞を作成することによって、より大きなフォトマスクを用いてスケールアップすることができます。増加した空洞の大きさは、両側のスペーサの幅を増加させることによって作成することができます。プロトコルでMNアレイを作製するために、各?...

経皮薬物送達は、特にほぼ独占的に皮下注射によって投与された生体分子のために、薬剤投与のための魅力的な代替的なアプローチを提供しています。しかし、皮膚、特に最上層（角質層）は、人体に侵入する外来性分子を防止手ごわい障壁です。最近では、MNのデバイスは、皮膚を介して薬物を送達するためのツールを有効にするとして浮上しています。 MNデバイスは、薬物分子の通過が改善された患者のコンプライアンスと利便1-3で所望の生理学的活性を達成できるように、角質層内部に一時的な空孔を作成します。 様々な製造方法は、ポリマーのMN 4を製造するために採用されています。しかし、それらは通常のMNのアレイを製造するために長い時間および/ ​​または高い温度を必要とする、複雑で多段階のプロセスを伴う。4製造工程を簡略化するために、単一工程モールドフリープロセスを使用してフォトマスクは5,6、最近開発されました。しかし、この方法では、メカニズムは、フォトリソグラフィに関係する紫外線（UV）光路を変更するための場所ではなかったとしてのMNは、鈍針のヒントを持っていた製作。 本研究では、フォトマスクに埋め込まれたマイクロレンズのMNのジオメトリを定義するために提案されています。埋め込まれたマイクロレンズからなり、その後、フォトマスクを用いて、鋭い先端が報告されていると製作をMNのフォトマスクを製造するためのプロトコル。

<table border="0" cellpadding="0" cellspacing="0">
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		<tr>
			<td>Poly(ethylene glycol) diacrylate (PEGDA Mn=258)</td>
			<td>SIGMA&#160;</td>
			<td>475629-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>2-hydroxy-2-methyl-propiophenone (HMP)</td>
			<td>SIGMA&#160;</td>
			<td>405655-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine collagen type 1, FITC conjugate&#160;</td>
			<td>SIGMA&#160;</td>
			<td>C4361</td>
			<td></td>
		</tr>
		<tr>
			<td>UV curing station&#160;&#160;&#160;</td>
			<td>EXFO Photonic Solutions Inc., Canada</td>
			<td>OmniCure S200-XL</td>
			<td></td>
		</tr>
		<tr>
			<td>Collimating Adaptor&#160;</td>
			<td>EXFO Photonic Solutions Inc., Canada</td>
			<td>EXFO 810-00042</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon SMZ 1500 stereomicroscope&#160;</td>
			<td>Nikon, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dillon GL-500 digital force gauge&#160;</td>
			<td>Dillon, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>A-1R confocal microscope&#160;</td>
			<td>Nikon, Japan</td>
			<td></td>
			<td></td>
		</tr>
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			<td></td>
			<td></td>
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polymeric microneedle array fabrication by photolithography

本稿では、フォトリソグラフィ法により、高分子マイクロニードル（MN）アレイの製造を説明しています。これは、埋め込まれたマイクロレンズからなるフォトマスクを用いて、単純な鋳型を含まないプロセスを含みます。埋め込まれたマイクロレンズは​​、MNジオメトリ（鋭さ）に影響することが見出されました。二つの異なる長さ（193ミクロンと171ミクロン±957ミクロン±1336ミクロン）を作製したと41.5ミクロン±8.4ミクロンと71.6ミクロン±13.7ミクロンの間の範囲の先端径の堅牢MNアレイ、。これらMNアレイは、皮膚を介して、低分子および高分子の治療薬の送達の潜在的なアプリケーションを提供してもよいです。

MNの形状は大きくフォトマスクの特性及び埋め込みマイクロレンズによって影響を受けることができます。屈折度は、MNの幾何形状（ 図2A）の影響紫外線の伝送路に影響を与えます。各マイクロレンズは350μmの直径を有することが見出された、130ミクロンの凸面を平坦化し、62.3μmの深さ（ 図2B-D）。ピタゴラスの定理を用いて、第1の表面の曲率半径は、272.89マイクロ...

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Polymeric Microneedle Array Fabrication by Photolithography

Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography.

フォトリソグラフィ法により高分子マイクロニードルアレイの作製

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a ...

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11.9K ビュー.  National University of Singapore. Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography. 

ビデオ: Polymeric Microneedle Array Fabrication by Photolithography

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

動的なマスク投影リソグラフィーを用いたニューラル培養システムのためのマイクロパターンハイドロゲルの作製

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

生物工学

Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale (nanoparticles). Nanoparticles can be engineered in a precise fashion where their size, composition and surface chemistry can be carefully controlled. This enables unprecedented freedom to modify some of the fundamental properties of their cargo, such as solubility, diffusivity, biodistribution, release characteristics and immunogenicity. Since their inception, nanoparticles have been utilized in many areas of science and medicine, including drug delivery, imaging, and cell biology1-4. However, it has not been fully utilized outside of "nanotechnology laboratories" due to perceived technical barrier. In this article, we describe a simple method to synthesize a polymer based nanoparticle platform that has a wide range of potential applications. 
The first step is to synthesize a diblock co-polymer that has both a hydrophobic domain and hydrophilic domain. Using PLGA and PEG as model polymers, we described a conjugation reaction using EDC/NHS chemistry5 (Fig 1). We also discuss the polymer purification process. The synthesized diblock co-polymer can self-assemble into nanoparticles in the nanoprecipitation process through hydrophobic-hydrophilic interactions.
The described polymer nanoparticle is very versatile. The hydrophobic core of the nanoparticle can be utilized to carry poorly soluble drugs for drug delivery experiments6. Furthermore, the nanoparticles can overcome the problem of toxic solvents for poorly soluble molecular biology reagents, such as wortmannin, which requires a solvent like DMSO. However, DMSO can be toxic to cells and interfere with the experiment. These poorly soluble drugs and reagents can be effectively delivered using polymer nanoparticles with minimal toxicity. Polymer nanoparticles can also be loaded with fluorescent dye and utilized for intracellular trafficking studies. Lastly, these polymer nanoparticles can be conjugated to targeting ligands through surface PEG. Such targeted nanoparticles can be utilized to label specific epitopes on or in cells7-10.

This article describes a nanoprecipitation method to synthesize polymer-based nanoparticles using diblock co-polymers. We will discuss the synthesis of diblock co-polymers, the nanoprecipitation technique, and potential applications.

ナノ析出法を通じて、ジブロック高分子ナノ粒子の製剤

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale ...

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective.
Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin.1-2 Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements.3-7,8
This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH,9 which are important metabolic indicators of disease states7,10-14 (e.g., cancer proliferation) and exercise-induced acidosis.15

This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors.

多重経皮電気化学センシングのための中空マイクロニードルベースの​​センサ

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

ナノ粒子トラッキング分析とハイスループットフローサイトメトリーによる高分子遺伝子デリバリーナノ粒子の評価

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

あらかじめ設計されたサイズと形状を用いるポリマー繊維およびバイオハイブリッド繊維のマイクロ流体製造

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

溶剤アシスト脂質二重（SALB）法による生体膜の作製

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

バイオメディカル応用のための超疎水性高分子材料を製造します

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

バルブ付き多層マイクロ流体デバイスのための多段階可変高さのフォトリソグラフィ

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

多重の人工細胞の微小環境配列の作製

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

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.