我々は、実験デザインとデータ分析の有用な議論のために、T. Shatovaに感謝します。 G·パラディの支援と専門知識は、マサチューセッツ工科大学のコッホ研究所のコアフローサイトメトリー、およびマイクロシステム技術研究所のスタッフの人が感謝して承諾されます。この作品は、健康補助金RC1 EB011187-02、DE013023、DE016516、EB000351の国立研究所によってサポートされ、部分的に国立がん研究所がんセンターサポート（コア）で、P30-CA14051およびMPP-09Call-ランガー-60を付与した。

1。ストレージ<ol><li> 70％エタノール中貯水池、ホルダー、Oリングやマイクロ流体デバイスに保管してください。ほこりや外部粒子による蒸発や汚染を防止するための蓋を持っているコンテナ（ 例えば瓶やビーカー）を使用します。 （3）第三の内の1つの容器（1）と、第2の容器内のリザーバとO-リング（2）と、ホルダー内のデバイスを置く。 </li></ol>注：ストレージのための70％エタノールの使用は、無菌性を維持することである。ソリューションのコンポーネントのみがエタノールと水（ すなわち無変性剤）がある場合は、すべてのシステム·コンポーネントは、完全に互換性がなければならず、時間の経過とともに低下することはありません。 <ol start="2"><li>実験にわたって交差汚染を防止し、目詰まりを起こすことができ、望ましくない粒子の存在を最小にするために、各使用前に、エタノール中の溶液の容器（2）と（3）を交換してください。 </li></ol> 2。実験の準備<ol><li> foの超音波浴に置き容器（2）及び（3）各使用前にR 5〜10分。これは、前の実験から任意の汚染粒子を除去するのに役立ちます。 </li><li> 70％エタノール溶液でバイオセーフティーキャビネット中できれいなワークスペース。 </li><li>生物学的安全キャビネット内に置く前に、70％エタノール溶液ですべての材料（3容器、及びピンセット）を噴霧する。 </li></ol> 3。アセンブリ<ol><li>作業領域に2〜3の低リントワイプ上下セット。 </li><li>ピンセットでそのコンテナからプラスチック製のリザーバーを取り外し、内面からのエタノール溶液の蒸発を促進するためにワイプ上に設定してください。穏やかに表面上のリザーバをタップまたはエタノール溶液の除去を容易にするために、それらを介して空気を吹き込む。 </li><li>貯水池上での適切なスロットにOリングを挿入します。 </li><li>それぞれの容器からホルダーとチップを取り外し、エタノールを蒸発させる（〜1〜2分）。 </li><li>ホルダーに必要なチップフェイスアップ（最大すなわちアクセスホール）を配置するためにピンセットを使用してください。ホルダーを上げる必ずチップをホルダーに平らに置かれていることを確認し、必要に応じてピンセットで調整するeyelevelにチップを搭載した。重要：デバイスがそのホルダ内に正しくフィットしない場合、それはその後の工程中に破損する危険性がある。 </li><li>次に、そっとホルダーに貯水池を置き、クリップでそれらを合わせます。 Oリングは、このプロセスの間に、そのスロットから落ちないように注意してください。 </li><li>彼らは、カチッと音がするまで静かに貯水池を下に押します。貯水池の両側は、セキュアチップが正しい位置にあるように見えるということであることを確認してください。 </li></ol> 4。細胞調製<ol><li>接着細胞（プライマリまたは確立された行）の場合：1〜2日前に、実験にプレート細胞彼らは実験日には80％以上コンフルエントないようにする。 </li><li> （PBSまたは関連するメディアに）懸濁液中の細胞を置き、1.0×10 6個/ ml 7 CEL 10 X 1.0へのオペレーティング濃度を目指すlsの個/ ml。注：我々は、セル濃度に配信性能に大きな変化は確認されていません。 </li><li>実験に必要な物質濃度を得るために、別のチューブに細胞や配送希望の材料を混ぜる。重要：記載の送達方法は、送達を容易にするために、拡散に依存しているため、より高い物質の濃度がより高い送達をもたらす。可能な場合は、最初の試験のために必要な材料の1μMのソリューションを使用することをお勧めします。必要に応じて、この濃度は、将来の実験でダウン滴定することができる。このデバイスに使用される最低報告された濃度は、10 nMで21です。 </li></ol> 5。操作<ol><li>ピペットリザーバ（現在の設計は、最大150μlの容量を有している）への細胞および送達物質との混合物。注：ほとんどのデバイスの設計は、そのためのチャネルを通る流れの方向は重要ではありません、完全に可逆的である。サンプルは、2つのリザーバのいずれかにロードされてもよい。 </李&gt; <li>満たされたタンクに圧力チューブを取り付け、（指のタイトが十分であることが多い）適切なシールを確保するために、ナットを締めます。 </li><li>レギュレータ上の所望のレベルに圧力を調整します。これは、細胞が装置を通って移動する速度を制御する。ボタンが押されるまで、セルの流れが開始されないことに注意してください。 </li></ol>注：前作では、窒素または圧縮空気をキャリアガスとしても同様に取り組んできました。 <ol start="4"><li>目の高さにデバイスを高め、リザーバ内の液体が簡単に見えるようにそれを向けます。注：貯水池が空になる前にシステムを遮断する液柱を追跡します。 </li><li>リザーバを加圧すると、セルの流れを開始するためにボタンを押してください。 </li><li>液面がリザーバの底部から約2mmである場合には、速やかに流れを停止させる0 psiまでレギュレータを向ける。重要：リザーバが空になる前に、1つの流れを停止しなかった場合、一方はcollectioから試料を排出するリスクn個の貯水池。また、流体カラム（ 例えば 3倍遅い）かなりのペースで移動していない、又は実質的にその初期流量に対して鈍化していることに注意した場合、車載器は、おそらく詰まると交換する必要がある。詰まったデバイスを動作させると、より高い細胞死につながることができます。 </li><li>適切なリザーバから処理された細胞を収集し、必要なコレクションチューブ/プレートに配置します。重要：porated細胞は最大10分20のための材料を取り込みしていきますように、この段階で任意のバッファに採取した細胞を希釈しないでください。このウィンドウが経過した後、所望の培地/緩衝液で細胞を希釈する。 </li><li>必要に応じて5.7 - より多くの処理された細胞を収集したり、代わりの実験条件を試してみてくださいするには、手順5.1を繰り返します。チップは可逆的であることを思い出して、そのための試料は、リザーバーのどちらかに取り付けることができます。彼らが目詰まりした場合、必要​​に応じてチップを交換してください。詰まったデバイスを廃棄します。 </li></ol> 6。解体<ol><li&gt;そっと脇にクリップアームを押してメインホルダーから貯水池を外します。 </li><li> （セクション1で詳述）適切な貯蔵容器内の各部品を配置します。 </li><li>廃棄のために別の容器に使用されるチップを置きます。 </li></ol>

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著者アーモンSharei、ロバート·ランガー、及びKlavs F·ジェンセンは、この記事で使用するマイクロ流体デバイスを製造SQZバイオテクノロジー会社の株主である。

記載の実験方法（チップ設計及び動作速度以外、すなわち因子）の特定の局面では、システムが適用される細胞タイプおよび送達材料に応じて最適化される必要があり得る。アドレスに実験を設計する際に考慮すべき最も一般的な要因のいくつかを以下の考察。 蛍光標識された化合物のための送出信号を改善するためには、バックグラウンド蛍光の供給源に対処?...

細胞の細胞質への巨大分子の送達は、治療および研究用途における重要なステップである。タンパク質送達は、臨床検査室3と4の両方の設定で細胞機能に影響を与える有望な手段であるが、ナノ粒子媒介送達は、例えば、遺伝子治療の1,2のポテンシャルを示している。そのような小分子薬物、量子ドット、金ナノ粒子などの他の材料は、5,6癌治療からの細胞内標識7,8、および9を追跡単一分子までの範囲の用途において重要である。 細胞膜は、巨大分子の大部分は不透過性である。多くの既存の技術が膜破壊やエンドサイトーシスの配信を容易にするために、ポリマーナノ粒子10,11、リポソーム12、または化学修飾13を使用しています。これらの方法では、送達効率および細胞生存率は、多くの場合、目の構造に依存する電子標的分子および細胞型。これらの方法は、核酸のような構造的に均一な材料の配達時に効率的になりますが、多くの場合、そのようなタンパク質14,15ナノ材料7など、より構造的に多様な材料の配信のために病気に適している。さらに、これらの方法のほとんどはに依存しているエンドソーム破壊メカニズムは、したがって、小胞構造16に捕捉された多くの素材を残し、多くの場合、非効率的である。最後に、多くの場合、確立された細胞株を使用するために開発されている方法は、一次細胞にうまく変換されません。 例えば、エレクトロポレーション17,18および19ソノポレーションのような膜ポレーション法は、いくつかの用途において魅力的な代替物であるが、それらは、低い細胞生存率を引き起こすことが知られており、標的デリバリー材料の電荷によって制限され得る。 細胞の急速な機械的変形、納品までのマイクロ流体アプローチは、最近になっていますdemonstr細胞の再プログラミング20とナノマテリアルの配信21のコンテキストで現在の技術に比べてその利点をated。この方法は、周囲の緩衝液中に存在する物質のサイトゾル送達を促進する細胞膜oを機械的な破壊に依存している。システムが以前に挑戦的細胞型において潜在的に可能にする（ 例えば、一次免疫細胞および幹細胞）を実証し、物質（ 例えば 、抗体およびカーボンナノチューブ）している。ここで、標的巨大分子の細胞内送達のためにこれらのデバイスを使用するための一般的な手順を説明する。しかし、それは以前に報告されていないアプリケーションのために、私たちの以前に報告された設計指針20で詳述するように1が、条件の簡単な最適化を実施することをお勧めします。手順は、ほとんどの細胞型および送達物質に一般化される。今日までに、システムは、RNA、DNA、金ナノ粒子、量子ドット、カーボンナノチューブ、タンパク質の送達のために首尾よく使用されているS、およびデキストランポリマー20,21。

<table border="0" cellpadding="0" cellspacing="0" width="777">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:375px;">Device Holder &#38; Plastic reservoir</td>
			<td style="width:136px;">SQZ Biotechnologies</td>
			<td style="width:149px;">Holder</td>
			<td style="width:117px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LSRFortessa Analyzer</td>
			<td>Becton Dickinson</td>
			<td>N/A</td>
			<td>Flow cytometry machine used at the Koch Institute Core Facilities</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microfluidic device</td>
			<td>SQZ Biotechnologies</td>
			<td>Cell Squeeze</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">O-Rings</td>
			<td>McMaster</td>
			<td>9452K311</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pressure system to operate device</td>
			<td>SQZ Biotechnologies</td>
			<td>Pressure System</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tweezers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ultrasound bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

cell squeezing as a robust, microfluidic intracellular delivery platform

細胞の急速な機械的な変形は高分子およびナノ物質の細胞内送達のための有望な、ベクトルフリー法として浮上している。この技術は、以前は困難な用途に対処する電位を示した;を含む一次免疫細胞、細胞の再プログラミング、カーボンナノチューブ、量子ドット送達への送達。このベクターを含まないマイクロ流体プラットフォームは、ターゲット材料のサイトゾル送達を容易にするために、細胞膜の機械的破壊に依存している。ここで、我々は、デバイス組み立て、細胞調製物、およびシステム動作を含むこれらのマイクロ流体デバイスのための使用の詳細な方法を記載している。このアプローチは、送達装置タイプ、以前に報告されていないアプリケーションの動作条件の簡単な最適化を必要とする。このシステムは、特殊な緩衝剤または化学修飾/コンジュゲーション工程を必要としないように提供される命令は、ほとんどの細胞型および送達物質に一般化される。この作品も提供Sデバイスの性能を向上させ、目詰まり、低配信効率、および細胞の生存率に関連する潜在的な問題をトラブルシュートする方法に関する推奨事項。

図1は、微小流体送達システムの概略的記述を含んでいる。 図2a〜図bは蛍光共役3 kDaのデキストラン20の存在下で、異なるデバイスの設計でHeLa細胞を処理することから、典型的な結果を示す。手順が正しく実行された場合、システム性能は、デバイスタイプと動作速度に敏感であろう。そのため、人はより複雑な実験に進む前に、特定のアプリケーションに?...

Watch this Scientific Journal Video about Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform at JoVE.com

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

細胞の急速な機械的な変形は高分子およびナノ物質の細胞内送達のための有望な、ベクトルフリー法として浮上している。このプロトコルは、幅広いアプリケーションのためのシステムを使用する方法の詳細な手順を提供しています。

堅牢、マイクロ流体細胞内送達プラットフォームとしての細胞絞る

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

cell-squeezing-as-robust-microfluidic-intracellular-delivery

Research

JoVE Journal

Bioengineering

12.7K ビュー.  Massachusetts Institute of Technology. 細胞の急速な機械的な変形は高分子およびナノ物質の細胞内送達のための有望な、ベクトルフリー法として浮上している。このプロトコルは、幅広いアプリケーションのためのシステムを使用する方法の詳細な手順を提供しています。 

ビデオ: Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

異なる系統にヒト胚性幹細胞（hESCの）の伝播との差別化のための3Dプラットフォームとしてのアルギン酸マイクロカプセル

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

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 ...

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments,&#160;as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier,&#160;more quantifiable extracellular relay of signals to the cells. 
For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

多臓器チップ - 長期マルチ組織の共培養のためのマイクロ流体プラットフォーム

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

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 ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

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

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

粒子の空間追跡のための多重化された電子の検出とマイクロ流体プラットフォーム

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

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved useful as a convenient live imaging platform providing capabilities for precise control of experimental conditions in real time. In this article, we demonstrate live imaging of individual worms employing WormSpa, a previously-published custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes. To illustrate the capability, we performed proof-of-principle experiments in which worms were infected in the device with pathogenic bacteria, and the dynamics of expression of immune response genes and egg laying were monitored continuously in individual animals. The simple design and operation of this device make it suitable for users with no previous experience with microfluidic-based experiments. We propose that this approach will be useful for many researchers interested in longitudinal observations of biological processes under well-defined conditions.

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In this article, we demonstrate live imaging of individual worms employing a custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes.

カエノラブディティス ・ エレガンスにおける縦断的イメージングのためのマイクロ流体プラットフォーム

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

組換えコラーゲン ペプチド Microcarriers 細胞伸長と細胞バイオリアクター配信システムとして潜在的な使用のためのモデルします。

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

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 ...

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

長期無細胞遺伝子発現の伝導のための多層マイクロ流体プラットフォーム

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...

A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth plate cartilage architecture and results in growth modulation of long bones of children. To determine the role of compressive stress in bone growth, we created a microfluidic device actuated by pneumatic pressure, to dynamically (or statically) compress growth plate chondrocytes embedded in alginate hydrogel cylinders. In this article, we describe detailed methods for fabricating and characterizing this device. The advantages of our protocol are: 1) Five different magnitudes of compressive stress can be generated on five technical replicates in a single platform, 2) It is easy to visualize cell morphology via a conventional light microscope, 3) Cells can be rapidly isolated from the device after compression to facilitate downstream assays, and 4) The platform can be applied to study mechanobiology of any cell type that can grow in hydrogels.

This article provides detailed methods for fabricating and characterizing a pneumatically actuating microfluidic device for chondrocyte compression.

動的圧縮で軟性細胞を刺激するマイクロ流体プラットフォーム

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth ...