Name: perfusable vascular network with a tissue model in a microfluidic device
Uploaded: 2018-04-04T14:00:00
Description: Watch this Scientific Journal Video about Perfusable Vascular Network with a Tissue Model in a Microfluidic Device at JoVE.com

Dette arbejde blev støttet af CREST JST (grant nummer JPMJCR14W4), samfund for at fremme af videnskab (JSP'ER) KAKENHI (grant nummer 25600060, 16K 16386), The Center for Innovation Program fra MEXT og JST, projekt fokuseret på at udvikle nøglen evaluering teknologi fra Japan Agency for medicinsk forskning og udvikling, AMED, Mizuho fundament til fremme af videnskab. Microfabrication blev støttet af Kyoto University Nano teknologi Hub.

1. fabrikation af formen mikrofluid enhed
<ol><li>Design mønster af mikrofluid enheden ved hjælp af kommercielt tilgængelige software (Clewin5 eller AutoCAD 2016, osv.). Funktionen af Clewin5, se brugsanvisning (http://manualzz.com/doc/7159150/clewin-user-s-manual). Bemærk: Filen design er tilgængelig i supplerende fil 1.</li>
	<li>Overføre filen design til en mikro mønster generator og indlæse værktøjet med en krom maske belagt med den positive photoresist.</li>
	<li>Udsætte den...

<ol>
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Tidligere rapporter viser, at hLFs udskiller en cocktail af flere angiogene faktorer, såsom angiopoietin-1, angiogenin, hepatocyt vækstfaktor, omdanne vækst faktor-α, tumor nekrose faktor og nogle ekstracellulære matrix proteiner29, 30. Denne analyse er afhængig af angiogene sekretion fra hLFs i en coculture sfæroide, der er begrænsningen i teknikken. Derfor er det afgørende for en stabil vaskulære formation til at fastsætte en coculture klumpformet i...

Flytte fra en éncellelag (todimensional) kultur til en tre-dimensionel kultur er motiveret af behovet for at arbejde med kultur-modeller, der efterligner de cellulære funktioner af levende væv1,2,3. Fladt og hårdt plast substrater almindeligt anvendt i cellekultur ligner ikke de fleste af de ekstracellulære miljøer i den menneskelige krop. I virkeligheden, viser mange undersøgelser, at tre-dimensionelle kultur genskabe væv-specifikke arkitektur, mekaniske og biokemiske stikord og cell-celle kommunikation, som ikke er blevet observeret i konventionelle to-dimensionelle kultur4, 5,6,7,8.
En flercellet aggregat eller sfæroide, er en af de mest lovende teknikker til at realisere denne tre-dimensionelle kultur9,10. Celler udskiller den ekstracellulære matrix (ECM) og kan kommunikere med andre i sfæroide. Selv om nogle andre bioteknologi tilgange11,12,13,14, såsom celle stabling, med held kopiere rumlige kompleksiteten af det menneskelige legeme, disse tilgange har kun to eller tre former for celler for lethed af analyse og fokuseret på kun én funktion af målorganer. Derimod udsættes celler i spheroids for forskellige kultur miljøer afhængig af deres positioner i klumpformet på grund af de heterogene levering af næringsstoffer, ilt, og paracrine og autocrine signalering molekyler i sfæroide. Denne funktion af spheroids efterligner delvist i vivo kultur tilstand og aktivere celler i spheroids at skabe langt mere komplekse, organiseret væv struktur i vitro end kulturperler i stabling væv9, 15 , 16. Bemærk, at hvis en klumpformet består af en enkelt slags celler, funktionen af cellerne i sfæroide ikke er ensartet på grund af de heterogene miljø i sfæroide. I de sidste par år, klumpformet kulturer tilladt embryonale stamceller (økonomiske), induceret pluripotente stamceller (iPSCs) eller væv-resident stamceller til at efterligne i vivo udviklingsmæssige sekvenser og genskabe mini-organer som hjerne17, leveren18og nyrerne19,20.
Trods betydelige fremskridt i klumpformet kultur teknikker er dyrkning store spheroids i lang tid stadig problematisk. I en tre-dimensionel væv skal celler være placeret inden for 150-200 µm i en blodåre på grund af det begrænsede udbud af ilt og næringsstoffer21. Vaskulære netværk inden for sfæroide er nødvendige for at sammenfatte udveksle stoffer mellem blod og væv in vivo. For at opnå dette, har andre grupper Co kulturperler endotelceller med target celler22,23,24 eller induceret pluripotente celler differentiering i CD31-positive celler20. De rapporterede fartøj-lignende strukturer har dog ikke de åbne ender af lumina at levere ilt og næringsstoffer til midten af sfæroide. For at efterligne den vaskulære rolle for at nære cellerne i de tre-dimensionelle kultur, skal åben og perfusable vaskulære netværk udvikles i sfæroide.
I løbet af de sidste par år, nogle forskningsgrupper i feltet microengineering rapporteret metoder til at konstruere en perfusable vaskulære netværk, spontant dannet i en mikrofluid enhed ved at udnytte angiogene faktorer fra cocultured fibroblastceller25 ,26. Disse vaskulære netværk har en lignende morfologi til deres i vivo modparter og kan være ombygget af miljømæssige faktorer, hvilket gør dem egnede til efterligne vaskulære funktioner i en klumpformet kultur. Formålet med denne protokol er at konstruere en perfusable vaskulære netværk i en sfæroide, ved hjælp af en mikrofluid platform27. Enhedens mikrofluid er ændret fra de tidligere rapporterede enhed25 , så en klumpformet kan indarbejdes. Ved at lede angiogene sekretion fra fibroblastceller i en klumpformet i endothelial celler i microchannels, angiogene spirer fra microchannels anastomosed med sfæroide og dannet en perfusable vaskulære netværk. Denne metode giver mulighed for en direkte levering af en lang række stoffer, som fluorescerende molekyler og mikrometer skala perler ind i indre af en sfæroide, som danner rammen for en langsigtet vævskultur med vaskulære netværk.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AutoCAD 2017</td>
			<td>Autodesk</td>
			<td>AutoCAD 2017</td>
			<td></td>
		</tr>
		<tr>
			<td>A chromium mask coated with AZP 1350.</td>
			<td>CLEAN SURFACE TECHNOLOGY</td>
			<td>CBL2506Bu-AZP</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro pattern generator</td>
			<td>Heidelberg</td>
			<td>uPG101</td>
			<td></td>
		</tr>
		<tr>
			<td>MF CD-26 developer</td>
			<td>Rohm and haas electronic materials</td>
			<td>-</td>
			<td>Developer in protocol 1.4</td>
		</tr>
		<tr>
			<td>S-Clean</td>
			<td>Sasaki Chemical</td>
			<td>S-24</td>
			<td>Chromium etchant in protocol 1.5</td>
		</tr>
		<tr>
			<td>Aceton</td>
			<td>Wako</td>
			<td>012-00343</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon Wafer</td>
			<td>Canosis</td>
			<td>SiJ-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin Coater</td>
			<td>MIKASA</td>
			<td>1H-D7</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexamethyldisilazane (HMDS)</td>
			<td>Tokyo Ohka Kogyo</td>
			<td>H0089</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 3050</td>
			<td>MicroChem</td>
			<td>-</td>
			<td>Negative photoresist in protocol 1.9</td>
		</tr>
		<tr>
			<td>UV Exposure</td>
			<td>Nanometric Technology Inc</td>
			<td>LA310s</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 Developer</td>
			<td>MicroChem</td>
			<td>Y020100</td>
			<td>Developer for the negative photoresist in protocol 1.13</td>
		</tr>
		<tr>
			<td>2-propanol</td>
			<td>Wako</td>
			<td>163-04841</td>
			<td></td>
		</tr>
		<tr>
			<td>Surhace profiler</td>
			<td>Vecco</td>
			<td>Veeco Dektak XT-S</td>
			<td></td>
		</tr>
		<tr>
			<td>(Trichloro (1H, 1H, 2H, 2H-perfluorooctyl) silane</td>
			<td>Sigma</td>
			<td>448931</td>
			<td></td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS)</td>
			<td>Dow Corning Toray</td>
			<td>184W/C</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy Punch (1.0mm)&#160;</td>
			<td>Kai Industries</td>
			<td>BP-10F</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy Punch (2.0mm)&#160;</td>
			<td>Kai Industries</td>
			<td>BP-20F</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma System</td>
			<td>Femto Science</td>
			<td>COVANCE</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>MATSUNAMI GLASS</td>
			<td>C024241</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture Dishes</td>
			<td>Iwaki</td>
			<td>1000-035</td>
			<td></td>
		</tr>
		<tr>
			<td>RFP Expressing Human Umbilical Vein Endothelial Cell</td>
			<td>Angio Proteomie</td>
			<td>cAP-0001RFP</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Human Lung Fibroblasts</td>
			<td>Lonza</td>
			<td>CC-2512</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth Medium</td>
			<td>Lonza</td>
			<td>CC-3162</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibroblast Growth Media Kits</td>
			<td>Lonza</td>
			<td>CC-3132</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution</td>
			<td>Wako</td>
			<td>168-23191</td>
			<td></td>
		</tr>
		<tr>
			<td>0.05w/v% Trypsin-0.53mmol/l EDTA&#8226; 4Na Solution with Phenol Red</td>
			<td>Wako</td>
			<td>204-16935</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (Phosphate Buffered Salts)</td>
			<td>Takara bio</td>
			<td>T900</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Sumitomo bakelite</td>
			<td>631-21031</td>
			<td></td>
		</tr>
		<tr>
			<td>1000ul Chip</td>
			<td>NIPPON Genetics</td>
			<td>FG-402</td>
			<td></td>
		</tr>
		<tr>
			<td>200ul&#160; Chip</td>
			<td>NIPPON Genetics</td>
			<td>FG-301</td>
			<td></td>
		</tr>
		<tr>
			<td>10ul Chip</td>
			<td>NIPPON Genetics</td>
			<td>37650</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td>Model 370</td>
			<td></td>
		</tr>
		<tr>
			<td>GFP Expressing Human Umbilical Vein Endothelial Cell</td>
			<td>Angio Proteomie</td>
			<td>cAP-0001GFP</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibrinogen from bovine plasma</td>
			<td>Sigma</td>
			<td>F8630</td>
			<td></td>
		</tr>
		<tr>
			<td>Aprotinin from bovine lung</td>
			<td>Sigma</td>
			<td>A6279</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen I</td>
			<td>Corning</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin from bovine plasma</td>
			<td>Sigma</td>
			<td>T4648</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Invitrogen</td>
			<td>H21492</td>
			<td>Fluorescent dye to stain nuclei in protocol 5.5</td>
		</tr>
		<tr>
			<td>Paraformaldehyde Solution</td>
			<td>Wako</td>
			<td>163-25983</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Fluorescence Microscope</td>
			<td>OLYMPUS</td>
			<td>IX71</td>
			<td></td>
		</tr>
		<tr>
			<td>Degital CCD Camera</td>
			<td>OLYMPUS</td>
			<td>ORCA-R2</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Laser Scanning Biological Microscope</td>
			<td>OLYMPUS</td>
			<td>FV1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Fluorescence Microscope</td>
			<td>OLYMPUS</td>
			<td>IX-83</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate-dextran</td>
			<td>Sigma</td>
			<td>FD70S</td>
			<td></td>
		</tr>
	</tbody>
</table>

perfusable vascular network with a tissue model in a microfluidic device

En klumpformet (en flercellet samlet) betragtes som en god model for levende væv i den menneskelige krop. Trods betydelige avancement i klumpformet kulturer forbliver en perfusable vaskulære netværk i spheroids en kritisk udfordring til langtidskulturer forpligtet til at opretholde og udvikle deres funktioner, såsom protein udtryk og morfogenese. Protokollen præsenterer en ny metode for at integrere en perfusable vaskulære netværk inden for klumpformet i en mikrofluid enhed. For at fremkalde en perfusable vaskulære netværk i sfæroide, blev angiogene spirer tilsluttet microchannels guidet til sfæroide ved at udnytte angiogene faktorer fra menneskelige lunge fibroblaster kulturperler i sfæroide. Angiogene spirer nåede sfæroide, fusioneret med i endothelial celler Co kulturperler i sfæroide, og dannede en kontinuerlig vaskulære netværk. Den vaskulære netværk kunne perfuse indre af sfæroide uden enhver lækage. Den beregnede vaskulære netværk kan anvendes yderligere som en rute til levering af næringsstoffer og fjernelse af affaldsprodukter, efterligne blod omløb in vivo. Metoden giver en ny platform i klumpformet kultur mod bedre sammenfatning af levende væv.

Figur 1 viser et design og foto af mikrofluid enheden. Det har tre parallelle kanaler, i hvilken kanal 2 indeholder sfæroide godt. Kanal 1 og 3 anvendes for HUVEC kulturen og kanal 2 er for sfæroide. Hver kanal er adskilt af trapezformet microposts designet til mønster PDMS. Microposts forhindre hydrogel i kanal 2 fra siver ud i kanal 1 og 3 af overfladespænding og give mulighed for udveksling af stoffer mellem klumpformet og HUVECs i microchannels<sup cl...

Watch this Scientific Journal Video about Perfusable Vascular Network with a Tissue Model in a Microfluidic Device at JoVE.com

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

Perfusable vaskulære netværk med en væv Model i en mikrofluid enhed

Protokollen beskriver, hvordan du ingeniør en perfusable vaskulære netværk i en klumpformet. Den sfæroide omkringliggende mikromiljø er udtænkt for at fremkalde angiogenese og forbinde sfæroide til microchannels i en mikrofluid enhed. Metoden tillader perfusion af den sfæroide, der er en længe ventet teknik i tre-dimensionelle kulturer.

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

Research

JoVE Journal

Bioengineering

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

A Microfluidic Device for Studying Multiple Distinct Strains

En mikrofluid enhed for at studere flere forskellige stammer

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

Mikrofluidisk fremstilling af polymere og biohybridfibre med foruddefineret størrelse og form

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Protokol for Biofilm Streamer Dannelse i en mikrofluidindretning med Micro-søjler

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

Analyserer Blanding Inhomogeniteter i en mikrofluidapparat af Individuel Schlieren Technique

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren ...

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

Microfluidic genipin Deposition Teknik for Extended Culture of mikromønstrede Vaskulære Muskuløse Thin Films

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

Custom-designed Laser-based Heating Apparatus for Triggered Release of Cisplatin from Thermosensitive Liposomes with Magnetic Resonance Image Guidance

Custom-designet laser-baserede Varme Apparater til Udløst Frigivelse af cisplatin fra varmefølsom Liposomer med magnetisk resonans billede Vejledning

Liposomes have been employed as drug delivery systems to target solid tumors through exploitation of the enhanced permeability and retention (EPR) effect ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

En Mikrofluid platform for High-throughput Single-celle Isolering og Kultur

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 ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Co-kultur af Living microbiome med Microengineered Menneskelig Intestinal Villi i en Gut-on-a-Chip mikrofluidanordning

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

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

Image-styret, Laser-baserede Fremstilling af vaskulær-afledte Mikrofluid Networks

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