我々は、この研究で利用さPFPE-PEG系界面活性剤の例については、レインダンスの技術に感謝し、我々はPDMSチャネルのレプリカを作成するために使用されるシリコンウェハモールド用バイオMEMSリソースセンター（メフメットトナー、ディレクター）に感謝。

<ol>
	<li>Zagnoni, M., Lain, G. L. e., Cooper, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrocoalescence+mechanisms+of+microdroplets+using+localized+electric+fields+in+microfluidic+channels.">Electrocoalescence mechanisms of microdroplets using localized electric fields in microfluidic channels.</a> Langmuir : the ACS journal of surfaces and colloids. 26, 14443-14449 (2010).</li><li>Niu, X. Z., Gielen, F., Edel, J. B., deMello, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microdroplet+dilutor+for+high-throughput+screening.">A microdroplet dilutor for high-throughput screening.</a> Nat. Chem. 3, 437-442 (2011).</li><li>Vincent, M. E., Liu, W., Haney, E. B., Ismagilov, R. 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JEは、この原稿で利用技術に基づく特許出願中の発明です。

発注の比較的高い程度にもかかわらず、すべてのドロップは、粒子または細胞の適切な数が含まれています。カプセル化効率は、その総数で割った望ましい占有と滴にカプセル化されたようになる細胞または粒子の数として計算することができる。これらの生データを収集し、エマルジョンのサンプル自動化された高速ビデオ·アルゴリズムから、またはイメージングのいずれかから取得することができます。これは、P kは k個の粒子とk個の粒子を含んでいるでしょうが値下がりしましたD kの画分を含むドロップダウンにカプセル化された粒子の割合と比較することができます。 図3から、両方のシングルとダブルの粒子のカプセル化効率は2倍以上で、ランダムなカプセル化の効率を上回る大幅粒子の所望の数より多いと液滴の数を減らす図4は、高効率のための適切な濃度の必要性を示しています。つまり、＆lambd<html> 、、粒子濃度とドロップボリュームの両方の機能は、正しくカプセル化粒子または細胞を最大限にするには、ドロップ当たり所望の細胞の数と等しいか、または近くに配置する必要があります。緻密な列車は時間の経過とともに広がると列車間のemptier領域を埋めるために傾向があるので、粒子または細胞の高濃度が完全に順序は通常良いことであることに注意してください。濃度が高すぎる一方で、粒子数が多いノズルで噴射誘導する界面不安定性を引き起こす可能性があります。特定の研究（例えば、単一セルのカプセル化、など）に、それはさらにいくつかの空の液滴を導入することを犠牲にして複数セルの液滴を避けるために、より有利かもしれないが、わずかに低くλが望まれるよう。また、これは2つの細胞間または細胞と単粒子または単一セルの液滴が細胞または粒子の一種の二つ以上の液滴よりも許容である粒子間の相互作用を目指した研究に適用されます。 jove_content &quot;&gt;時間をかけて定数λを維持することは一貫性のあるカプセル化のために重要です。シリンジおよびチューブ内の細胞と粒子の沈降を減少させることにより長期的濃度制御の浮力のマッチングを支援します。しかし、浮力が高い水溶液粘度でも結果に一致する可能性（長い焦点チャネルの要件になる）発注遅延、チャネルの圧力降下を増加させ、ドロップ生成に必要な流量を変更します。この実験で使用される一致浮力の代わりに、物理的に注射器のコンセントが指摘されているようにシリンジポンプを傾けることであるほぼ垂直に下向き（シリンジ内部に細胞または粒子の付着を最小限に抑えるため）。ここでは、1.3％（mL当たり約25万粒子）の粒子の体積分率で9.9μmの直径のミクロスフェアを使用しましたが、我々は、体積分率を高めるために傾斜を利用図3に示すように、データの2％に第二の選択肢は水性流体intermittを混在させることです。ently小さな外部磁石を使用して、同封のステンレススチールボールベアリング（テフロンは、細胞を操作するためのコーティング）である。ケアは、それが入口管への入り口をふさぐ可能性のあるボールベアリングは、注射器の先端に定住させることを避けるためにただし、必要があります。しかし、これらの選択肢はより多くの労働集約的と浮力のマッチング未満の再現性があるので、浮力の照合では、長いタイムフレーム上で発生する大規模な実験に最も適しています。一方、慣性順序は、水性及び油の流れが定常滴14を噴射になります（ 図2を参照）、制御されていないカプセル化結果の滴り、高く高く押されたときに、高い更新を必要とし、動作するようにpを再。ここで使用さ10μmの粒子よりも小さいセルの場合、小さいチャネルの寸法は、流量を噴射することなく増加させることができない場合は、十分な再p を達成するために必要な場合があります。マイクロ流体システムで噴射のいずれかの特性は、ヒステリシスの影響がwを発生することができるということですHICHが難しく、それが観察されなかったポイントに戻って発生した後、単に水の流量を下げることによって噴射を停止するように。一つは、以前にドロップ発生率の追加の輪郭は、ドロップ当たりの細胞と軸方向の共同流れるノズル14とT-ジャンクション26から28用に開発されているようなフローマップを噴射する水滴次元または無次元を開発することができ、実験結果に基づいており、カプセル化効率。このマップは、ドロップの発生率はλ計算するため、水と油の流れ事前の推定流量を提供するために予測することができ、そこから強力なロードマップを提供するであろう。 直接ここに示されていませんが、 図3bに示されているものからの油の流量Q 油の追加削減はさらにその上に3つ、4つのドロップ当たりの粒子数を増やす、となります。ドロップあたりの粒子を達成するために、いずれかのQ 油が減少またはaqu必要がありますeous流量Q AQは増やす必要があります。余談ですが、私たちはオンラインで補足含まれている<a href="http://www.jove.com/files/ftp_upload/4096/4096_drop_encapsulation.m" target="_blank">MATLABスクリプト</a>滴の粒子の任意の数を捕捉するの封入効率をモデル化します。ユーザは、入力の平均粒子間隔と順序の度合いをモデル粒子間隔の標準偏差を、。注文した列車については、標準偏差は小さくなります。さらに、ユーザーの入力の平均滴径とドロップサイズドロップサイズの多分散性を占める標準偏差、。追加情報については、スクリプトのマニュアルを参照してください。 ときに水流量を増やしたり、極端な値の近くに、それぞれの流量として不安定な噴射の増加のリスクを低下当たりの粒子または細胞の数を増やすためのオイル流量を減少させる。従って、達成可能な粒子/ドロップ当たりのセルの最大数デバイスの形状と流体特性に依存します。粒子/細胞濃度と油の流量を考えると、ドロップ当たりの粒子/細胞の数は、水性の流量の上限によって制限され、順序を誘導するのに十分な大きさでなければなりませんが、噴射不安定を避ける（とせん断を制限するために十分小さくなければならない可能性を確保するために細胞のストレス）。また、順序が発生した水の流量を考えると、油の流量が滴り政権に残っているのに十分な十分な大きさでなければなりません。 ドロップダウンの生成と遷移を噴射する水滴は界面活性剤濃度に非常に敏感であることに注意してください。高い界面活性剤の濃度は、ドロップ生成パラメータを変更して、油の粘度を増加させます。余談ですが、フッ素系油の広く利用可能な生体適合性界面活性剤の不足は大きな課題を提示します。現在、一つの商業サプライヤー（レインダンステクノロジーズ）は、PFPE-PEGブロック共重合体界面活性剤、2のために存在して5しかし、研究はそのようなPFPE-HEGとして界面活性剤·グループの数の小規模な合成技術を実証する。このような光の鉱物油として29,30の代替は、使用可能な界面活性剤の広い範囲、24,31にアクセスするための生物学的なドロップ生成アプリケーションで利用されているしかし、フッ素油に比べて粘度の増加が付随するドロップ生成パラメータを変化させることに注意してください。最近のレビュー32は公開され、連続相の油と界面活性剤の多数を説明しています。

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td> AutoCADの</td><td>オートデスク</td><td></td><td></td></tr><tr><td>透明マスク</td><td>のFineLineイメージング株式会社</td><td></td><td></td></tr><tr><td> SU-8フォトレジスト</td><td>マイクロケム</td><td> 2050 </td><td></td></tr><tr><td> Dektakプロフィル</td><td> Veeco社</td><td></td><td></td></tr><tr><td>ペトリ皿</td><td> BDファルコン</td><td> 351058 </td><td></td></tr><tr><td> PDMSシリコーンエラストマーキット</td><td>ダウコーニング（株） </td><td> Sylgard 184、材質番号（240）4019862 </td><td></td></tr><tr><td>減圧デシケーター</td><td> Jencons </td><td> 250から030 </td><td></td></tr><tr><td>真空ポンプ</td><td>アルカテル·バキューム·テクノロジー</td><td> 2010 C2 </td><td></td></tr><tr><td>真空レギュレータ</td><td>コー​​ル·パーマー</td><td> EW-00910から10 </td><td></td></tr><tr><td>オーブン</td><td>サーモフィッシャーサイエンティフィック</td><td>リンドバーグブルーM、OV800F </td><td></td></tr><tr><td>生検パンチ、0.75ミリメートル</td><td>ハリス</td><td>ユニコア15072 </td><td></td></tr><tr><td>実験室コロナ処理機</td><td>エレクトロ·テクニック·プロダクツ株式会社</td><td> BD-20AC、SKU 12051A </td><td></td></tr><tr><td>スライドガラス</td><td>ゴールドシール</td><td> 3010 </td><td></td></tr><tr><td> Aquapel </td><td> PPGインダストリーズ</td><td></td><td>代替戦略</td></tr><tr><td>ポリスチレンマイクロスフェア、9.9μmの</td><td>サーモ</td><td> G1000 </td><td></td></tr> &lt;TR&gt; <td> OptiPrep </td><td> Sigma-Aldrich社</td><td> D1556 </td><td>示されていません</td></tr><tr><td>ルアーロックシリンジ</td><td> BD </td><td> 1 ML：309628 3 ML：309585 </td><td></td></tr><tr><td> FC-40フッ素オイル</td><td> 3M社</td><td>シグマアルドリッチ、F9755 </td><td></td></tr><tr><td> PFPE-PEGのフッ素系界面活性剤</td><td>レインダンステクノロジーズ</td><td></td><td></td></tr><tr><td>軽油</td><td> PTIプロセスケミカル</td><td> 08042-47-5 </td><td>代替戦略</td></tr><tr><td>ミネラルオイルの界面活性剤</td><td>エボニックゴールドシュミット社</td><td> ABIL EM 90 </td><td>代替戦略</td></tr><tr><td>タイゴンPVCチューブ</td><td> SmallParts </td><td> TGY-010 </td><td></td></tr><tr><td> 30ゲージルアーロックシリンジニードル、1/2 &quot; </td><td> SmallParts </td><td> NE-301PL-C </td><td></td></tr><tr><td>倒立顕微鏡</td><td>カールツァイスイメージング</td><td>アクシオObserver.Z1 </td><td></td></tr><tr><td>ハイスピー​​ドカメラ</td><td>ビジョン研究</td><td>ファントムV310 </td><td></td></tr><tr><td>シリンジポンプ（2） </td><td> Chemyx株式会社</td><td>ネクサス3000 </td><td></td></tr><tr><td>シリコーンオイル</td><td>ダウコーニング</td><td> 200流体、10 cStの</td><td>エマルジョンストレージ用のオプション</td></tr></tbody></table>

high throughput single-cell and multiple-cell micro-encapsulation

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 &mu;m polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency Pk=1 of 83.7% and a double-particle encapsulation efficiency Pk=2 of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed. 
Introduction 
Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify signals from bioreactor products. Drops also provide the ability to re-merge drops into larger aqueous samples or with other drops for intercellular signaling studies.1,2 The reduction in dilution implies stronger detection signals for higher accuracy measurements as well as the ability to reduce potentially costly sample and reagent volumes.3 Encapsulation of cells in drops has been utilized to improve detection of protein expression,4 antibodies,5,6 enzymes,7 and metabolic activity8 for high throughput screening, and could be used to improve high throughput cytometry.9 Additional studies present applications in bio-electrospraying of cell containing drops for mass spectrometry10 and targeted surface cell coatings.11 Some applications, however, have been limited by the lack of ability to control the number of cells encapsulated in drops. Here we present a method of ordered encapsulation12 which increases the demonstrated encapsulation efficiencies for one and two cells and may be extrapolated for encapsulation of a larger number of cells.
To achieve monodisperse drop generation, microfluidic "flow focusing" enables the creation of controllable-size drops of one fluid (an aqueous cell mixture) within another (a continuous oil phase) by using a nozzle at which the streams converge.13 For a given nozzle geometry, the drop generation frequency f and drop size can be altered by adjusting oil and aqueous flow rates Qoil and Qaq. As the flow rates increase, the flows may transition from drop generation to unstable jetting of aqueous fluid from the nozzle.14 
When the aqueous solution contains suspended particles, particles become encapsulated and isolated from one another at the nozzle. For drop generation using a randomly distributed aqueous cell suspension, the average fraction of drops Dk containing k cells is dictated by Poisson statistics, where Dk = &lambda;k exp(-&lambda;)/(k!) and &lambda; is the average number of cells per drop. The fraction of cells which end up in the "correctly" encapsulated drops is calculated using Pk = (k x Dk)/&Sigma;(k' x Dk'). The subtle difference between the two metrics is that Dk relates to the utilization of aqueous fluid and the amount of drop sorting that must be completed following encapsulation, and Pk relates to the utilization of the cell sample. As an example, one could use a dilute cell suspension (low &lambda;) to encapsulate drops where most drops containing cells would contain just one cell. While the efficiency metric Pk would be high, the majority of drops would be empty (low Dk), thus requiring a sorting mechanism to remove empty drops, also reducing throughput.15
Combining drop generation with inertial ordering provides the ability to encapsulate drops with more predictable numbers of cells per drop and higher throughputs than random encapsulation. Inertial focusing was first discovered by Segre and Silberberg16 and refers to the tendency of finite-sized particles to migrate to lateral equilibrium positions in channel flow. Inertial ordering refers to the tendency of the particles and cells to passively organize into equally spaced, staggered, constant velocity trains. Both focusing and ordering require sufficiently high flow rates (high Reynolds number) and particle sizes (high Particle Reynolds number).17,18 Here, the Reynolds number Re =uDh/&nu; and particle Reynolds number Rep =Re(a/Dh)2, where u is a characteristic flow velocity, Dh [=2wh/(w+h)] is the hydraulic diameter, &nu; is the kinematic viscosity, a is the particle diameter, w is the channel width, and h is the channel height. Empirically, the length required to achieve fully ordered trains decreases as Re and Rep increase. Note that the high Re and Rep requirements (for this study on the order of 5 and 0.5, respectively) may conflict with the need to keep aqueous flow rates low to avoid jetting at the drop generation nozzle. Additionally, high flow rates lead to higher shear stresses on cells, which are not addressed in this protocol. The previous ordered encapsulation study demonstrated that over 90% of singly encapsulated HL60 cells under similar flow conditions to those in this study maintained cell membrane integrity.12 However, the effect of the magnitude and time scales of shear stresses will need to be carefully considered when extrapolating to different cell types and flow parameters. The overlapping of the cell ordering, drop generation, and cell viability aqueous flow rate constraints provides an ideal operational regime for controlled encapsulation of single and multiple cells.
Because very few studies address inter-particle train spacing,19,20 determining the spacing is most easily done empirically and will depend on channel geometry, flow rate, particle size, and particle concentration. Nonetheless, the equal lateral spacing between trains implies that cells arrive at predictable, consistent time intervals. When drop generation occurs at the same rate at which ordered cells arrive at the nozzle, the cells become encapsulated within the drop in a controlled manner. This technique has been utilized to encapsulate single cells with throughputs on the order of 15 kHz,12 a significant improvement over previous studies reporting encapsulation rates on the order of 60-160 Hz.4,15 In the controlled encapsulation work, over 80% of drops contained one and only one cell, a significant efficiency improvement over Poisson (random) statistics, which predicts less than 40% efficiency on average.12
In previous controlled encapsulation work,12 the average number of particles per drop &lambda; was tuned to provide single-cell encapsulation. We hypothesize that through tuning of flow rates, we can efficiently encapsulate any number of cells per drop when &lambda; is equal or close to the number of desired cells per drop. While single-cell encapsulation is valuable in determining individual cell responses from stimuli, multiple-cell encapsulation provides information relating to the interaction of controlled numbers and types of cells. Here we present a protocol, representative results using polystyrene microspheres, and discussion for controlled encapsulation of multiple cells using a passive inertial ordering channel and drop generation nozzle.

Watch this Scientific Journal Video about High Throughput Single-cell and Multiple-cell Micro-encapsulation at JoVE.com

High Throughput Single-cell and Multiple-cell Micro-encapsulation

細胞と粒子の慣性順序を持つ単分散ドロップ生成を組み合わせることで、我々は、kHzのレートで一滴の細胞または粒子の所望の数をカプセル化する方法を説明します。我々は、二回のシングルとダブルの粒子降下を順不同カプセルのものを超えて効率を示しています。

ハイスループットマイクロカプセル化シングルセルと複数セル

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement ...

high-throughput-single-cell-and-multiple-cell-micro-encapsulation

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

18.6K ビュー.  Vanderbilt University. 細胞と粒子の慣性順序を持つ単分散ドロップ生成を組み合わせることで、我々は、kHzのレートで一滴の細胞または粒子の所望の数をカプセル化する方法を説明します。我々は、二回のシングルとダブルの粒子降下を順不同カプセルのものを超えて効率を示しています。

ビデオ: High Throughput Single-cell and Multiple-cell Micro-encapsulation

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

バイオマテリアルのハイスループット検出用高分子マイクロアレイ

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

生物工学

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

マイクロ流体プラットフォームを使用した高スループットのタンパク質発現ジェネレータ

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with conventional one-sample-at-a-time synthesis techniques, a more recent high-throughput approach has been developed enabling the synthesis and testing of large libraries of polyanhydrides1. This will facilitate more efficient optimization and design process of these biomaterials for drug and vaccine delivery applications. The method in this work describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries. In this robotically operated method (Figure 1), linear actuators and syringe pumps are controlled by LabVIEW, which enables a hands-free automated protocol, eliminating user error. Furthermore, this method enables the rapid fabrication of micro-scale polymer libraries, reducing the batch size while resulting in the creation of multivariant polymer systems. This combinatorial approach to polymer synthesis facilitates the synthesis of up to 15 different polymers in an equivalent amount of time it would take to synthesize one polymer conventionally. In addition, the combinatorial polymer library can be fabricated into blank or protein-loaded geometries including films or nanoparticles upon dissolution of the polymer library in a solvent and precipitation into a non-solvent (for nanoparticles) or by vacuum drying (for films). Upon loading a fluorochrome-conjugated protein into the polymer libraries, protein release kinetics can be assessed at high-throughput using a fluorescence-based detection method (Figures 2 and 3) as described previously1. This combinatorial platform has been validated with conventional methods2 and the polyanhydride film and nanoparticle libraries have been characterized with 1H NMR and FTIR. The libraries have been screened for protein release kinetics, stability and antigenicity; in vitro cellular toxicity, cytokine production, surface marker expression, adhesion, proliferation and differentiation; and in vivo biodistribution and mucoadhesion1-11. The combinatorial method developed herein enables high-throughput polymer synthesis and fabrication of protein-loaded nanoparticle and film libraries, which can, in turn, be screened in vitro and in vivo for optimization of biomaterial performance.

This method describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries.

組合せの合成と高分子膜とナノ粒子のライブラリからのハイスループットタンパクリリース

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with ...

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1. Several biodegradable polymers have been studied as vaccine delivery vehicles1; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12.
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17.
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

無水物ナノ粒子のハイスループット糖鎖の合成と機能化

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

High Throughput Microfluidic Rapid and Low Cost Prototyping Packaging Methods

In this work, 3 different packaging and assembly techniques are presented. They can be classified into two categories: one-time use and reusable packaging techniques.
The one-time use packaging technique employs UV-based and temperature curing epoxies to connect microtubes to access holes, wire-bonding for integrated circuit connections, and silver epoxy for electrical connections. This method is based on a robust assembly technique that can support relatively high pressure close to 1 psi and does not need any support to strengthen the microfluidic architecture.
Reusable packaging techniques consist of PDMS-based microtube interconnectors and anisotropic adhesive films for electrical connections. These devices are more sensitive and fragile. Consequently, Plexiglas support is added to the microfluidic structure to improve the electrical contact when anisotropic adhesive films are used, and also to strengthen the microfluidic architecture. In addition, a micromanipulator is needed to maintain tubes while using a thin PDMS layer to connect them to the access holes. Different PDMS layer thicknesses, ranging from 0.45-3 mm, are tested to compare the best adherence versus injection rates. Applied injection rates are varied from 50-300 &#956;l/hr for 0.45-3 mm PDMS layers, respectively. These techniques are mainly applicable for low-pressure applications. However, they can be extended for high-pressure ones through plasma-oxygen process to permanently seal the PDMS to glass substrates. The main advantage of this technique, besides the fact that it is reusable, consists of keeping the device observable when the microchannel length is very short (in the range of 3 mm or lower).

In this article we describe different techniques for microfluidic rapid prototyping platforms. The proposed techniques are based on ultraviolet (UV) sensitive and temperature curing epoxies, polydimethylsiloxane (PDMS) based tubing, wire-bonding, and anisotropic adhesive films. The assembling procedures presented are developed for both one-time use devices as well as reusable microfluidic systems.

ハイスループットマイクロ流体の迅速かつ低コストのプロトタイピング包装方法

In this work, 3 different packaging and assembly techniques are presented. They can be classified into two categories: one-time use and reusable packaging ...

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III secretion system (T3SS) in Shigella flexneri minicells. It can be applicable to any project suitable for cryo-ET.

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

Tomoautoの使用：ハイスループット自動クライオ電子断層撮影のためのプロトコル

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

ハイスループットシングルセルの単離および培養のためのマイクロ流体プラットフォーム

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as contributing to antimicrobial resistance. One possible avenue for the reduction of device-associated infections is the identification of bacteria-repellent polymer coatings for these devices, which would prevent bacterial binding at the initial attachment step. A method for the identification of such repellent polymers, based on the parallel screening of hundreds of polymers using a microarray, is described here. This high-throughput method resulted in the identification of a range of promising polymers that resisted binding of various clinically relevant bacterial species individually and also as multi-species communities. One polymer, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)), demonstrated significant reduction in attachment of a number of hospital isolates when coated onto two commercially available central venous catheters. The method described could be applied to identify polymers for a wide range of applications in which modification of bacterial attachment is important.

A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

医療機器のための細菌撥ポリマーのハイスループット同定

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

注射の再生療法と高スループットの薬剤のスクリーニングのための 3 D への放射能

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

単一細胞のゲノムの配列のための超高スループット マイクロ流体プラットフォーム

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...