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
Summary
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.
Abstract
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.
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Coverslip staining rack | Electron Microscopy Sciences | www.emsdiasum.com/ | 72239-04 | Alternative coverslip rack may be used |
| Microscope cover glass – 25 mm | Fisher Scientific, Inc. | www.fishersci.com | 12-545-102 | Alternative brand and size may be used; Microscope slides may also be substituted as substrate base |
| Poly(N-iso-propylacrylamide) (PIPAAm) | Polysciences, Inc. | www.polysciences.com/ | #21458 | Sigma-Aldrich makes an alternate compound, but we have not tested it for use with this protocol; Compound gives strong odor, use proper ventilation |
| 1-butanol | Sigma-Aldrich | www.sigmaaldrich.com | 360465 | Hazard: flammable (store stock solution in flammable cabinet); flash point is 37 °C, avoid heating; alternative product may be used |
| Spincoater | Specialty Coating Systems, Inc. | www.scscoatings.com | SCS G3P8 Model; Alternative brand and/or model may be used | |
| Polydimethylsiloxane (PDMS) | Ellsworth Adhesives (Dow Corning) | www.ellsworth.com | 184 SIL ELAST KIT 0.5KG | Alternative distributor may be used |
| Fluorescent microbeads | Polysciences, Inc. | www.polysciences.com/ | 17151 | Alternative brand and/or larger size may be used |
| Silicon wafers | Wafer World, Inc. | www.waferworld.com | 2398 | Alternative brand and/or size may be used |
| Photoresist | MicroChem Corp. | www.microchem.com | SU-8 3025 allows 20-25-µm feature height | |
| Contact mask aligner | Suss MicroTec | www.suss.com | MA6 contact mask aligner; alternative brand and/or model may be used for wafer exposure | |
| Developer | MicroChem Corp. | www.microchem.com | SU-8 Developer; Hazard: flammable | |
| Tridecafluro-trichlorosilane | UCT Specialties, Inc. | www.unitedchem.com | T2492 | Silane for non-stick coating of patterned silicon wafers (CAUTION: Tridecafluro-trichlorosilane is a flammable and corrosive liquid. Proper personal protective equipment and local exhaust is necessary for use. ) |
| Surgical biopsy punch | Integra LifeSciences Corp. | www.miltex.com | 33-31AA-P/25 | Alternative brand and/or size may be used |
| Genipin | Cayman Chemical | www.caymanchem.com | 10010622 | Sigma-Aldrich (G4796-25MG) makes an alternate compound, but we have not tested it for use with this protocol |
| 1X phosphate buffered saline | Mediatech, Inc. | www.cellgro.com | 21-031-CV | Alternative brand may be used |
| Fibronectin | Corning, Inc. | www.corning.com | 356008 | Sigma-Aldrich (F1056) makes an alternate compound, but we have not tested it for use with this protocol |
| Penicillin/streptomycin | Life Technologies, Inc. | www.lifetechnologies.com | 15140-122 | Alternative brand and/or size may be used, as long as concentration is the same |
| Umbillical artery smooth muscle cells | Lonza | www.lonza.com | CC-2579 | Alternative cell types may be used for alternative applications. Media should be modified accordingly |
| Tyrode's solution components | Sigma-Aldrich | www.sigmaaldrich.com | various | Alternative brand may be used for mixing solution |
| Stereomicroscope | Zeiss | www.zeiss.com | 4350020000000000 | SteREOLumar V12; Alternative brand/type of stereomicroscope may be used |
| Temperature-controlled platform | Warner Instruments | www.warneronline.com | 641659; 640352; 641922 | |
| Endothelin-1 | Sigma-Aldrich | www.sigmaaldrich.com | E7764-50UG | Alternative amount may be purchased, as long as treatment concentration is maintained |
| HA-1077 | Sigma-Aldrich | www.sigmaaldrich.com | H139-10MG | Alternative amount may be purchased, as long as treatment concentration is maintained |
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