Name: microfluidic genipin deposition technique for extended culture of micropatterned vascular muscular thin films
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Description: Watch this Scientific Journal Video about Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films at JoVE.com

We acknowledge financial support from the American Heart Association Scientist Development Grant, 13SDG14670062 (PWA) and the University of Minnesota Doctoral Dissertation Fellowship (ESH). We also acknowledge the microfabrication resources of the Minnesota Nano Center (MNC) and the image processing resources of the University Imaging Centers (UIC), both at the University of Minnesota. Parts of this work were carried out in the Characterization Facility, University of Minnesota, which receives partial support from NSF through the MRS program.

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The authors have nothing to disclose.

ここでは、慢性血管疾患経路1,23,24の拡張実験時間は、より一般的なことができ、以前に開発VMTF技術に基づいて構築されたプロトコルを提示します。これを達成するために、我々は、以前にMTF収縮実験で使用するための改良された血管組織の生存率で操作動脈ラメラを得るために、マイクロ流体堆積技術を用いて、PDMS基板11の長期的な官能化を提供することが示されているゲニピンを、微細。マケインら 。関連MTFモデル25に数週間のための工学的心臓組織の拡張文化のための代替micromoldedゼラチンハイドロゲル基材を開発しました。 このプロトコルは、正常に2週間にわたって動脈ラメラ構造（図2）と機能（図3）模倣vMTFsを得ました。提示されたプロトコルが正常に完了し、所望の解像度が得られているが疾患関連の時間経過の拡張培養時間のULT（ 例えば、脳血管攣縮の病理学的効果は14日まで26持続する）、いくつかの一般的な落とし穴が生じます。デバイス内の分岐の部分的または完全な閉塞をもたらし得る有害な損傷でPDMSマイクロ流体デバイス結果の繰り返し使用。したがって、新しいデバイスは、各実験のために使用されなければなりません。もう一つの問題は、のような一般的なものではないが、組織の予期せぬ矛盾剥離です。これらの発生は、発生中の自然の中でランダムで稀であるように思われました。観察により、組織を剥離する前に、主〜または内腔のような構造を形成します。また、マイクロコンタクト印刷技術を用いて製造組織におけるこの現象を観察しました。したがって、我々はこの問題はオーバーシーディングや異常行動ではなく、ここで紹介する製作方法の直接的な結果をもたらす培養したVSMCにおける表現型のスイッチのいずれかの結果であると考えています。 現在のマイクロ流体IC設計は、直線流パターンを必要とするような、単一の主方向に配向を有する組織に限定されます。このような心室の心筋構造27の「れんが壁」パターンなど、より複雑なパターン形成を必要とする組織への適用は、より複雑なマイクロ流体設計が必要になります。我々は、他の細胞型とゲニピン変性基質を接種していません。しかし、Genchi ら 。ゲニピン変性PDMS 11に等方性の骨格筋の拡張生存率を示しました。従って、我々は、このプロトコルの最小限に修正されたバージョンは、臓器·オン·チップ技術の将来の発展における他の器官系に広範囲の適用性を持っていることを確信します。 以前の方法に基づいて、8 vMTFsための応力計算方法は、組織の相対的な収縮及び薄膜の対応する切断長さによって制限されます。長すぎるカットした場合、フィルム自体にカールします。短すぎるカットした場合、フィルムはされませんND。いずれの場合には制限をモデルに起因する収縮特性を適切に分析することを防止します。適切な切断長さは、繰り返し実験により洗練されるべきです。 Agarwalさんらのように、レーザー彫刻システムの実装は、。、28を切断MTFの再現性と品質を改善することができます。 より良い厳重に管理、自己完結型の実験系では天然の組織の構造と機能を再現する能力は、生物工学の分野で重要な追求です。この追求は、基本的な生理学的および器官系29,30の病理学的挙動の理解を進め、いくつかのin vitroでの組織を模倣し、多機能、まだ単純化モデル器官オンチップの開発につながっています。 PDMS基板上ゲニピンの堆積を用いて、2週間の拡張、細胞の生存率と血管平滑筋機能の維持を実証しています。これは、以前の製造TECHNを大幅に改善されています文化19で7日、およびより堅牢な動脈オンチップ方法の開発を助けることができる- 4の後の組織および細胞死の剥離につながることができiques、。原因ほとんどの血管疾患の慢性的性質のために、この進歩は、特定の血管の病態に関与する収縮メカニズムに将来の調査の様々なフレームワークを提供します。

血管のような脳血管攣縮1,2、3高血圧、アテローム性動脈硬化症および4のような疾患、徐々に開発、自然界に一般的に慢性的であり、血管平滑筋細胞（VSMC）の機能不全の力の生成を伴います。我々は、in vivoモデルよりも実験条件をより詳細に制御を用いたin vitroの方法で使用して、これらの遅い進行血管機能障害を研究することを目指しています。我々は以前のin vitroの機能的収縮を測定するために、血管、筋肉薄膜（vMTFs）を開発した心血管組織5を設計しますが、この方法は、比較的短期間の研究に限定されていました。ここでは、長期的な測定のために私たちの前のVMTF技術を拡張基板修正手法を提示します。 内皮は、全体の血管機能でも重要であるが、人工動脈ラメラは、血管の変化を評価するための有用なモデルシステムを提供します疾患の進行中に収縮。機能性血管疾患の組織モデルを設計するために、動脈ラメラの構造と機能、血管の収縮基本ユニットの両方が、忠実に再現されている必要があります。動脈ラメラ、エラスチン6のシートによって分離された収縮のVSMCの円周方向に整列されたシート同心です。ポリジメチルシロキサン（PDMS）基板上に細胞外マトリックス（ECM）タンパク質のマイクロコンタクト印刷は、予め整列心臓血管組織5,7-10を模倣するために、組織の組織のガイダンスキューを提供するために使用されています。しかし、マイクロコンタクト印刷を使用してパターン化組織が慢性試験でその適用を制限し、培養液中の3-4日後に整合性を失う可能性があります。このプロトコルは、新しいマイクロ流体堆積技術を用いて、前のマイクロコンタクト印刷技術を置き換えることによって、この問題に対する解決策を提供します。 Genchi らゲニピンとfにPDMS基板を改変ound文化11で1月に筋細胞の生存率を延長しました。ここでは、PDMS上にパターン血管平滑筋細胞の培養物を拡張するために同様のアプローチを使用します。ゲニピン、クチナシ果実の自然加水分解誘導体は、同様の架橋剤および組織修復12,13とECMの変更14の分野における生体材料としての使用の増加に比べ、その比較的低い毒性のために、基板の修正のための望ましい候補であります15。このプロトコルでは、フィブロネクチンは、前のマイクロコンタクトプリント法のように、細胞のガイダンスキューとして利用されます。しかしながら、ゲニピンは、フィブロネクチンのパターニングの前にPDMS基板上に堆積されます。細胞がパターン化されたマトリックスを分解するとしてこのように、添付のVSMCから新たに合成されたECMは、ゲニピンでコーティングされたPDMS基板に結合することができます。 このプロトコルは、2段階ゲニピンとECM沈着のためのマイクロ流体送達装置を利用します。マイクロ流体デバイスの模倣体の設計microco以前の研究16において操作動脈ラメラに使用ntact印刷パターン。従って、我々は、このプロトコルが正常に生体内の構造と動脈ラメラの収縮機能に高度に整列を再現動脈ラメラ模倣を得ことを期待しています。我々はまた、ゲニピンは、インビトロ血管疾患モデルにおける長期的に適した基板修飾化合物であることを確認するために、組織の収縮性を評価します。

<table><tbody><tr><td>Coverslip staining rack</td><td>Electron Microscopy Sciences</td><td>&lt;a href=&quot;http://www.emsdiasum.com/&quot;&gt;www.emsdiasum.com/&lt;/a&gt;</td><td>72239-04</td></tr><tr><td>Microscope cover glass - 25 mm</td><td>Fisher Scientific, Inc.</td><td>&lt;a href=&quot;http://www.fishersci.com/&quot;&gt;www.fishersci.com&lt;/a&gt;</td><td>12-545-102</td></tr><tr><td>Poly(N-iso-propylacrylamide) (PIPAAm)</td><td>Polysciences, Inc.</td><td>&lt;a href=&quot;http://www.polysciences.com/&quot;&gt;www.polysciences.com/&lt;/a&gt;</td><td>#21458</td></tr><tr><td>1-butanol</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;www.sigmaaldrich.com&lt;/a&gt;</td><td>360465</td></tr><tr><td>Spincoater</td><td>Specialty Coating Systems, Inc.</td><td>&lt;a href=&quot;http://www.scscoatings.com/&quot;&gt;www.scscoatings.com&lt;/a&gt;</td><td></td></tr><tr><td>Polydimethylsiloxane (PDMS)</td><td>Ellsworth Adhesives (Dow Corning)</td><td>&lt;a href=&quot;http://www.ellsworth.com/&quot;&gt;www.ellsworth.com&lt;/a&gt;</td><td>184 SIL ELAST KIT 0.5KG</td></tr><tr><td>Fluorescent microbeads</td><td>Polysciences, Inc.</td><td>&lt;a href=&quot;http://www.polysciences.com/&quot;&gt;www.polysciences.com/&lt;/a&gt;</td><td>17151</td></tr><tr><td>Silicon wafers</td><td>Wafer World, Inc.</td><td>&lt;a href=&quot;http://www.waferworld.com/&quot;&gt;www.waferworld.com&lt;/a&gt;</td><td>2398</td></tr><tr><td>Photoresist&amp;nbsp;</td><td>MicroChem Corp.</td><td>&lt;a href=&quot;http://www.microchem.com/&quot;&gt;www.microchem.com&lt;/a&gt;</td><td></td></tr><tr><td>Contact mask aligner</td><td>Suss MicroTec</td><td>&lt;a href=&quot;http://www.suss.com/&quot;&gt;www.suss.com&lt;/a&gt;</td><td></td></tr><tr><td>Developer</td><td>MicroChem Corp.</td><td>&lt;a href=&quot;http://www.microchem.com/&quot;&gt;www.microchem.com&lt;/a&gt;</td><td></td></tr><tr><td>Tridecafluro-trichlorosilane</td><td>UCT Specialties, Inc.</td><td>&lt;a href=&quot;http://www.unitedchem.com/&quot;&gt;www.unitedchem.com&lt;/a&gt;</td><td>T2492</td></tr><tr><td>Surgical biopsy punch</td><td>Integra LifeSciences Corp.</td><td>&lt;a href=&quot;http://www.miltex.com/&quot;&gt;www.miltex.com&lt;/a&gt;</td><td>33-31AA-P/25</td></tr><tr><td>Genipin</td><td>Cayman Chemical</td><td>&lt;a href=&quot;http://www.caymanchem.com/&quot;&gt;www.caymanchem.com&lt;/a&gt;</td><td>10010622</td></tr><tr><td>1X phosphate buffered saline</td><td>Mediatech, Inc.</td><td>&lt;a href=&quot;http://www.cellgro.com/&quot;&gt;www.cellgro.com&lt;/a&gt;</td><td>21-031-CV</td></tr><tr><td>Fibronectin</td><td>Corning, Inc.</td><td>&lt;a href=&quot;http://www.corning.com/&quot;&gt;www.corning.com&lt;/a&gt;</td><td>356008</td></tr><tr><td>Penicillin/streptomycin</td><td>Life Technologies, Inc.</td><td>&lt;a href=&quot;http://www.lifetechnologies.com/&quot;&gt;www.lifetechnologies.com&lt;/a&gt;</td><td>15140-122</td></tr><tr><td>Umbillical artery smooth muscle cells</td><td>Lonza</td><td>&lt;a href=&quot;http://www.lonza.com/&quot;&gt;www.lonza.com&lt;/a&gt;</td><td>CC-2579</td></tr><tr><td>Tyrode&#039;s solution components</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;www.sigmaaldrich.com&lt;/a&gt;</td><td>various</td></tr><tr><td>Stereomicroscope</td><td>Zeiss</td><td>&lt;a href=&quot;http://www.zeiss.com/&quot;&gt;www.zeiss.com&lt;/a&gt;</td><td>4350020000000000</td></tr><tr><td>Temperature-controlled platform</td><td>Warner Instruments</td><td>&lt;a href=&quot;http://www.warneronline.com/&quot;&gt;www.warneronline.com&lt;/a&gt;</td><td>641659; 640352; 641922</td></tr><tr><td>Endothelin-1</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;www.sigmaaldrich.com&lt;/a&gt;</td><td>E7764-50UG</td></tr><tr><td>HA-1077</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;www.sigmaaldrich.com&lt;/a&gt;</td><td>H139-10MG</td></tr></tbody></table>

microfluidic genipin deposition technique for extended culture of micropatterned vascular muscular thin films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular behaviors on disease-relevant time scales. Previously, microcontact printing has been used to fabricate two-dimensional functional arterial mimics through patterning of extracellular matrix protein as guidance cues for tissue organization. Vascular muscular thin films utilized these mimics to assess functional contractility. However, the microcontact printing fabrication technique used typically incorporates hydrophobic PDMS substrates. As the tissue turns over the underlying extracellular matrix, new proteins must undergo a conformational change or denaturing in order to expose hydrophobic amino acid residues to the hydrophobic PDMS surfaces for attachment, resulting in altered matrix protein bioactivity, delamination, and death of the tissues.
Here, we present a microfluidic deposition technique for patterning of the crosslinker compound genipin. Genipin serves as an intermediary between patterned tissues and PDMS substrates, allowing cells to deposit newly-synthesized extracellular matrix protein onto a more hydrophilic surface and remain attached to the PDMS substrates. We also show that extracellular matrix proteins can be patterned directly onto deposited genipin, allowing dictation of engineered tissue structure. Tissues fabricated with this technique show high fidelity in both structural alignment and contractile function of vascular smooth muscle tissue in a vascular muscular thin film model. This technique can be extended using other cell types and provides the framework for future study of chronic tissue- and organ-level functionality.

この研究の主な目的は、疎水性のPDMS基板上にマイクロパターンのVSMCの生存を延長することでした。これは、PDMS（図1）上にパターンゲニピンおよびフィブロネクチンを堆積させるために、マイクロ流体送達システムを組み込むことによって達成されました。マイクロ流体送達を用いECMタンパク質の沈着は、ゲニピンおよびフィブロネクチン（図1D）のライン間の裸のPDMSとのチャネルパターンの忠実度の高い転送を得ました。以前のマイクロコンタクトプリント法5,10,16と同様の動脈ラメラのインビボ構造を模倣付着した細胞（図1E）フォームコンフルエントな単層（ 図2）。これらの組織は、その応力VMTF技術を用いて測定した（図3）に応答して、収縮構造を、得ました。 2週間にわたって組織の生存率の定性的評価は、ゲニピンMODIFIに最小限の劣化を示しました編基板（図2A）。組織合流とアライメントは二週間（図2B、2Cおよび2D）で維持しました。二つの重要な応力値は、すべてのVMTFについて計算した：1）基礎トーンと2）誘導された収縮（図3Cおよび3D）基礎トーンが平衡状態で刺激されていないのVSMCによって維持応力です。誘導された収縮はエンドセリン-1での刺激により誘導される追加の応力です。基礎トーンおよび誘導収縮の両方が（図3Eおよび3F）を通して血管作用組織を実証し、2週間の時間経過にわたって一貫性の挙動を示しました。血清飢餓のVSMCは、19を増殖しないので、アッセイの終了時に組織収縮のわずかな低下は、組織を構成する細胞の数の減少の直接の結果です。培養培地への血清の最小の基礎レベルの追加は、今後の作業では、この結果を軽減することができます。 <imgALT = &quot;図2&quot; SRC = &quot;/ファイル/ ftp_upload / 52971 / 52971fig2.jpg&quot; /&gt; 図2.組織が ​​正常にゲニピン変性基板上の二週間ミミックのインビボ動脈ラメラ構造生存したまま2週間の過程を通して犠牲の時点で組織の（A）代表位相コントラスト像（スケールバー：200ミクロン）。 F-アクチンフィラメント、青：核（細胞の存在を確立するために示されている）、スケールバー血清飢餓（緑後（B）1日目に固定ゲニピン変性基板上に作製した組織の代表的な免疫組織化学画像、4日目、および10日目。。 - 7）（D）組織アライメントF-アクチン配向秩序パラメータ（OOPによって測定される）22（エラーバー標準誤差、n = 3の：F-アクチンのカバレッジ（エラーバーにより測定さ100μm）（C）パーセントコンフルエンス：標準誤差、nは3から7）。 ©I​​OP PublishiNG。再現および/または許可を変更しました。全著作権所有。すべての権利を保有。19は、 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52971/52971fig2large.jpg" target="_blank">この図の拡大版を表示するには、こちらをクリックしてください。</a> <img alt="図3" src="/files/ftp_upload/52971/52971fig3.jpg" /> 図3.組織収縮は、2週間のコースにわたって維持されます。 （A）代表VMTF切断方式。（B）カットVMTFの模式図。 32℃以下に冷却すると、PIPAAmはVMTFを解放する、溶解します。アクティブセル層内の応力は、曲げにパッシブPDMS層を引き起こします。突出長さの測定は、Grosbergにおける方法に従って曲率半径に変換することができるら曲率半径8は、組織内の平均断応力を計算するために使用されている。（C）連続送信LIG代表的な収縮アッセイのHT画像（スケールバー：1ミリメートル）。組織が平衡に達し、完全な緩和を可能にするために、HA-1077で処理し、その後、エンドセリン1で刺激します。ボトム：収縮測定のためのアッセイの過程で理想化された組織の側面図の概略図（D）代表応力曲線。二つの主要な応力値を算出します。基礎音が平衡応力状態でリラックスした応力状態との差です。誘導された収縮状態をエンドセリン-1の平衡状態からの応力の変化刺激される（E）基礎トーン（エラーバー：標準誤差、nは5から12まで）。。（F）誘発性収縮（エラーバー：標準誤差、 N = 5から12）。 ©I​​OP出版。再現および/または許可を変更しました。すべての著作権は。19 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52971/52971fig3large.jpg" target="_blank">の拡大版を表示するには、こちらをクリックしてください</a>この図。

Watch this Scientific Journal Video about Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films at JoVE.com

Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films

We present a method for microfluidic deposition of patterned genipin and fibronectin on PDMS substrates, allowing extended viability of vascular smooth muscle cell-dense tissues. This tissue fabrication method is combined with previous vascular muscular thin film technology to measure vascular contractility over disease-relevant time courses.

マイクロパターン血管筋薄膜の拡張文化ためのマイクロ流体ゲニピン堆積技術

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular ...

microfluidic-genipin-deposition-technique-for-extended-culture

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

7.6K ビュー.  University of Minnesota. We present a method for microfluidic deposition of patterned genipin and fibronectin on PDMS substrates, allowing extended viability of vascular smooth muscle cell-dense tissues. This tissue fabrication method is combined with previous vascular muscular thin film technology to measure vascular contractility over disease-relevant time courses.

ビデオ: Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

機械的にダイナミックな細胞培養のための微細加工プラットフォーム

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

生物工学

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 ...

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

血液疾患における血管相互作用を研究するためのEndothelializedマイクロフルイディクス

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, and observation and quantification are very challenging. However, conventional in vitro microvessel assays have limitations when representing in vivo microvessels with respect to three-dimensional (3D) geometry and providing continuous fluid flow. Using a combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics, we have developed a multi-depth circular cross-sectional endothelialized microchannels-on-a-chip, which mimics the 3D geometry of in vivo microvessels and runs under controlled continuous perfusion flow. A positive reflowable photoresist was used to fabricate a master mold with a semicircular cross-sectional microchannel network. By the alignment and bonding of the two polydimethylsiloxane (PDMS) microchannels replicated from the master mold, a cylindrical microchannel network was created. The diameters of the microchannels can be well controlled. In addition, primary human umbilical vein endothelial cells (HUVECs) seeded inside the chip showed that the cells lined the inner surface of the microchannels under controlled perfusion lasting for a time period between 4 days to 2 weeks.

A microchannels-on-a-chip platform was developed by the combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics. The endothelialized microchannels platform mimics the three-dimensional (3D) geometry of in vivo microvessels, runs under controlled continuous perfusion flow, allows for high-quality and real-time imaging&#160;and can be applied for microvascular research.

マルチ深円形の断面内皮マイクロチャンネル·オン·ア·チップの開発のための手順

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

Microtissue安定化のためのマイクロ流体デバイスの層ごとのコラーゲン沈着

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

微細と3D微小血管の組立

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ- and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

血管由来のマイクロ流体ネットワークの画像誘導、レーザーベースの試作

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

マイクロ流体工学バイオプリンティング血管組織や Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

マイクロ流体デバイスにおける組織モデルと perfusable の血管ネットワーク

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

Microfluidic Model to Mimic Initial Event of Neovascularization

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the initial stage varies dramatically from the following process of neovascularization. Although there are plenty of models to simulate different stages of neovascularization, an in vitro 3D model that capitulates the initial process of neovascularization under the corresponding stimulations of normal vasculature microenvironments is still lacking. Here, we reconstructed an in vitro 3D model that mimics the initial event of neovascularization (MIEN). The MIEN model contains a microfluidic sprouting chip and an automatic control, highly efficient circulation system. A functional, perfusable microchannel coated with endothelium was formed and the process of sprouting was simulated in the microfluidic sprouting chip. The initially physiological microenvironment of neovascularization was recapitulated with the microfluidic control system, by which ECs would be exposed to high luminal shear stress, physiological transendothelial flow, and various vascular endothelial growth factor (VEGF) distributions simultaneously. The MIEN model can be readily applied to the study of neovascularization mechanism and holds a potential promise as a low-cost platform for drug screening and toxicology applications.

Here, we provide a microfluidic chip and an automatically controlled, highly efficient circulation microfluidic system that recapitulates the initial microenvironment of neovascularization, allowing endothelial cells (ECs) to be stimulated by high luminal shear stress, physiological level of transendothelial flow, and various vascular endothelial growth factor (VEGF) distribution simultaneously.

新血管新生初期事象を模倣する微発性モデル

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the ...