Microfluidic genipina Deposizione Tecnica per la cultura estesa di micropatterned vascolari muscolari Thin Films
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
Malattie vascolari, come il vasospasmo cerebrale 1,2, ipertensione 3, 4 e aterosclerosi, si sviluppano lentamente, sono tipicamente di natura cronica, e coinvolgere disfunzionale forza-generazione da parte delle cellule muscolari lisce vascolari (VSMC). Il nostro obiettivo è di studiare queste disfunzioni vascolari lenta progressione che utilizzano metodi in vitro con un controllo più preciso delle condizioni sperimentali rispetto ai modelli in vivo. Film Abbiamo già sviluppato vascolari muscolari sottili (vMTFs) per la misurazione della contrattilità funzionale in vitro progettati tessuti cardiovascolari 5, ma questo metodo è stata limitata a relativamente studi a breve termine. Qui vi presentiamo una tecnica modifica substrato che espande la nostra precedente tecnica vMTF per misure a lungo termine.
Mentre l'endotelio è fondamentale anche in funzione vascolare generale, lamelle arteriosa progettato fornire un sistema modello utile per valutare i cambiamenti in vascolarecontrattilità durante la progressione della malattia. Per progettare un modello di tessuto malattia vascolare funzionale, sia la struttura e la funzione della lamella arteriosa, l'unità contrattile base del recipiente, deve essere riassunta con alta fedeltà. Arteriosa lamelle sono concentrici, fogli circonferenzialmente allineati di VSMC contrattili separati da fogli di elastina 6. Stampa microcontact di matrice extracellulare (ECM) proteine su polidimetilsilossano (PDMS) substrati è stata precedentemente utilizzata per fornire spunti di orientamento per l'organizzazione dei tessuti di imitare allineato tessuto cardiovascolare 5,7-10. Tuttavia, i tessuti modellati utilizzando la stampa microcontact può perdere integrità dopo 3-4 giorni di cultura, limitando la loro applicabilità in studi cronici. Questo protocollo fornisce una soluzione a questo problema, sostituendo tecniche di stampa microcontact precedenti con una nuova tecnica di deposizione microfluidica.
Modificati PDMS substrati con genipina e f Genchi et al.ound prolungata sopravvivenza dei miociti fino a un mese di cultura 11. Qui, usiamo un approccio simile per estendere la cultura delle cellule muscolari lisce vascolari modellate su PDMS. Genipina, un derivato idrolitica naturale della frutta gardenia, è un candidato desiderabile per la modifica del substrato a causa della sua tossicità relativamente bassa rispetto ad agenti reticolanti simili e il suo uso crescente come biomateriale nel campo della riparazione tissutale 12,13 e ECM modifica 14, 15. In questo protocollo, la fibronectina è utilizzata come spunto di orientamento delle cellule, come in metodi di stampa microcontact precedenti; tuttavia, genipina viene depositata su PDMS substrati prima della fibronectina patterning. Così, come le cellule si degradano la matrice fantasia, ECM di nuova sintesi di VSMC collegati può legarsi al substrato PDMS genipina rivestite.
Questo protocollo utilizza un dispositivo di somministrazione microfluidica per due fasi genipina e ECM deposizione. Il design della microfluidica imita dispositivo microcomodelli di stampa ntact utilizzati per lamelle arteriosa progettato in studi precedenti 16. Quindi, ci aspettiamo che questo protocollo di cedere imita lamelle arteriosa che ricapitolano successo altamente allineato per struttura vivo e la funzione contrattile del lamelle arteriosa. Si valutano anche contrattilità tessuto per confermare che genipina è un composto modifica substrato adatto a lungo termine in vitro modelli di malattia vascolare.
Protocol
Representative Results
Discussion
Qui, vi presentiamo un protocollo che si basa sulla tecnologia vMTF precedentemente sviluppato, consentendo tempi prolungati esperimento più tipico dei percorsi croniche malattie vascolari 1,23,24. Per realizzare ciò, si micropattern genipina, che è stato precedentemente dimostrato di fornire funzionalizzazione a lungo termine di substrati PDMS 11, utilizzando una tecnica di deposizione microfluidi cedere lamelle arteriosa progettato con migliorata viabilità tessuto vascolare per esperimenti co…
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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