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.
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.
Vaskulære sygdomme, såsom cerebral vasospasme 1,2, hypertension 3, og atherosklerose 4, udvikler sig langsomt, er typisk kronisk karakter og involverer dysfunktionel kraft generation af vaskulære glatte muskelceller (VSMC). Vi sigter mod at studere disse langsomt fremadskridende vaskulære dysfunktioner anvender in vitro-metoder med finere kontrol af eksperimentelle betingelser end i in vivo modeller. Vi har tidligere udviklet vaskulære muskulære tynde film (vMTFs) til måling funktionel kontraktilitet af in vitro-manipuleret kardiovaskulære væv 5, men denne metode har været begrænset til relativt kortvarige studier. Her præsenterer vi et substrat modifikation teknik, der udvider vores tidligere vMTF teknik til langsigtede målinger.
Mens endotel er også kritisk i den samlede vaskulær funktion, manipuleret arteriel lameller giver en nyttig model system til at vurdere ændringer i vaskulærkontraktilitet under sygdomsprogression. At konstruere et funktionelt vaskulær sygdom vævsmodellen, både struktur og funktion af den arterielle lameller, den grundlæggende kontraktile enhed af fartøjet skal gengivet med high fidelity. Arteriel lamellerne er koncentriske, periferisk-aligned plader af kontraktile VSMC adskilt af plader af elastin 6. Microcontact trykning af ekstracellulær matrix (ECM) proteiner på polydimethylsiloxan (PDMS) substrater har tidligere været brugt til at give vejledning stikord til væv organisation at efterligne justeret kardiovaskulære væv 5,7-10. Men væv mønstrede hjælp microcontact udskrivning kan miste integriteten efter 3-4 dage i kultur, hvilket begrænser deres anvendelighed i kroniske studier. Denne protokol giver en løsning på dette problem ved at erstatte tidligere mikrokontakt trykteknikker med en ny microfluidic deposition teknik.
Genchi et al. Modificerede PDMS substrater med genipin og found langvarig levedygtighed myocytter op til en måned i kultur 11. Her bruger vi en lignende fremgangsmåde til at udvide kultur mønstrede vaskulære glatte muskelceller på PDMS. Genipin, en naturlig hydrolytisk derivat af gardenia frugt, er en ønskelig kandidat til substrat modifikation på grund af sin relativt lave toksicitet sammenlignet med lignende tværbindingsmidler og dens stigende anvendelse som et biomateriale på områderne vævsreparation 12,13 og ECM modifikation 14, 15.. I denne protokol, er fibronektin anvendes som en celle vejledning cue, som i tidligere mikrokontakt trykmetoder; imidlertid genipin afsat på PDMS substrater forud for fibronectin mønsterdannelse. Således som celler nedbryder den mønstrede matrix, kan nysyntetiserede ECM fra vedhæftede VSMC binde til genipin-coatede PDMS substrat.
Denne protokol udnytter en mikrofluid afgivelsesindretning til to-trins genipin og ECM deposition. Udformningen af mikrofluidapparatet efterligner microcoNTACT trykning mønstre anvendes til manipuleret arteriel lameller i tidligere undersøgelser 16. Således forventer vi denne protokol til at give arteriel lameller efterligner, der med held rekapitulere højt justeret in vivo struktur og kontraktile funktion af arteriel lameller. Vi evaluerer også væv kontraktilitet at bekræfte, at genipin er et egnet substrat modifikation sammensatte for langsigtet in vitro vaskulære sygdomsmodeller.
Her præsenterer vi en protokol, der bygger på tidligere udviklet vMTF teknologi, der giver udvidede eksperiment gange mere typisk for kroniske karsygdom veje 1,23,24. For at opnå dette, vi micropattern genipin, som tidligere er blevet vist at give langsigtet funktionalisering af PDMS substrater 11, under anvendelse af en mikrofluid deposition teknik til opnåelse manipuleret arteriel lameller med forbedret karvæv levedygtighed til brug i MTF kontraktilitet eksperimenter. McCain et al. …
The authors have nothing to disclose.
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.
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 |