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 arbeidet ble støttet av CREST JST (bevilgning nummer JPMJCR14W4), Society for det fremme av vitenskap (JSPER) KAKENHI (bevilgning nummer 25600060, 16K 16386), The Center of innovasjon Program fra MEXT og JST, prosjektet fokusert på utvikling nøkkelen evaluering teknologi fra Japan byrå for medisinsk forskning og utvikling, AMED, Mizuho Foundation for fremme av vitenskap. Microfabrication ble støttet av Kyoto University Nano teknologien Hub.

1. fabrikasjon av Microfluidic enheten Mold
<ol><li>Design mønster av microfluidic enheten bruker kommersielt tilgjengelig programvare (Clewin5 eller AutoCAD 2016, etc.). Hvis funksjonen av Clewin5, kan du se brukerhåndboken (http://manualzz.com/doc/7159150/clewin-user-s-manual). Merk: Filen design er tilgjengelige i supplerende fil 1.</li>
	<li>Overføre filen design til en mikro mønster generator og laste verktøyet med en krom maske belagt med den positive photoresist.</li>
	<li>Utset...

<ol>
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Tidligere rapporter viser at hLFs skiller en cocktail av flere angiogenic faktorer, slik som angiopoietin-1, angiogenin, hepatocyte vekstfaktor, omforming vekst faktor-α, tumor nekrose faktor og noen ekstracellulær matrix proteiner29, 30. Denne analysen er avhengig av angiogenic utskillelsen fra hLFs i en coculture-formet, som er begrensning av teknikken. Derfor er det avgjørende for en stabil vaskulær formasjon å sette en coculture-formet nederst på en sl...

Skifte fra en monolayer (todimensjonal) kultur til en tredimensjonal kultur er motivert av behovet for å arbeide med kultur modeller som etterligner de cellulære funksjonene levende vev1,2,3. Flat og hard plast underlag brukte i cellekultur ligner ikke de fleste av de ekstracellulære miljøene i menneskekroppen. Faktisk viser mange studier at tredimensjonale kultur gjenopprette vev-spesifikke arkitektur, mekaniske og biokjemiske signaler og celle-celle kommunikasjon, som ikke er observert i konvensjonelle todimensjonal kultur4, 5,6,7,8.
En flercellet samlet eller spheroid, er en av de mest lovende teknikkene for å realisere denne tredimensjonale kultur9,10. Celler skiller den ekstracellulære matrisen (ECM) og kan kommunisere med andre i formet. Selv om noen andre bioteknologi tilnærminger11,12,13,14, som cellen stabling, gjenskape romlige kompleksiteten av menneskekroppen, disse metodene har to eller tre typer celler for enkel analyse og fokusert på bare en funksjon av målet organer. Derimot utsatt celler i spheroids for annen kultur miljøer avhengig av sine posisjoner i spheroid på grunn av heterogene tilførselen av næringsstoffer, oksygen, og paracrine og autocrine signalnettverk molekyler i formet. Denne funksjonen i spheroids etterligner delvis i vivo kultur tilstand og Aktiver cellene i spheroids å skape mye mer kompleks, organisert vevet struktur i vitro enn kultivert i stabling vev9, 15 , 16. Hvis en spheroid består av en enkelt type celler, funksjonen til cellene i spheroid er ikke ensartet på grunn av heterogene miljø i formet. I de siste årene, formet kulturer tillatt embryonale stamceller (ESCs), indusert pluripotent stamceller (iPSCs) eller vev-resident stilk celler å etterligne i vivo utviklingsmessige sekvenser og gjenskape mini-organer som hjernen17, leveren18og nyre19,20.
Til tross for betydelige fremskritt i spheroid kultur teknikker er dyrking store spheroids lenge fortsatt problematisk. I en tredimensjonal vev må celler være plassert innenfor 150-200 µm av en blodåre på grunn av den begrensede tilgangen på oksygen og næringsstoffer21. Vaskulære nettverk innenfor spheroid er nødvendig å recapitulate utveksle stoffer mellom blod og vev i vivo. For å oppnå dette, har andre grupper co kultivert endotelceller med målet cellene22,23,24 eller indusert differensiering av pluripotent celler i CD31-positive celler20. Rapporterte fartøy-lignende strukturer har imidlertid ikke de åpne endene lumina å levere oksygen og næringsstoffer til midten av formet. For å etterligne vaskulær rollen ernære celler i tredimensjonale kultur, må åpent og perfusable vaskulære nettverket utvikles i formet.
De siste årene, noen forskningsgrupper i feltet microengineering rapportert metoder for å konstruere et perfusable vaskulære nettverk, spontant dannet i en microfluidic-enhet ved å benytte angiogenic faktorer fra cocultured fibroblast celler25 ,26. Disse vaskulære nettverk har en lignende morfologi for motpartene i vivo og kan bli ombygd av miljømessige faktorer, gjør dem egnede til å etterligne vaskulær funksjoner i en spheroid kultur. Formålet med denne protokollen er å bygge en perfusable vaskulære nettverket i en spheroid bruker en microfluidic plattform27. Microfluidic enheten er endret fra tidligere rapportert enheten25 slik at en spheroid kan innlemmes. Regi angiogenic utskillelsen fra fibroblast celler i en spheroid til endotelceller i microchannels, angiogenic spirer fra microchannels anastomosed med formet og dannet en perfusable vaskulære nettverk. Denne metoden gir en direkte levering av en rekke stoffer, som fluorescerende molekyler og mikrometer skala perler i indre av en spheroid, som inneholder fundamentet for en langsiktig vev kultur med vaskulære nettverk.

<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>
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		<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 spheroid (en flercellet samlet) regnes som en god modell av levende vev i kroppen. Til tross for betydelige fremme i spheroid kulturer fortsatt en perfusable vaskulære nettverket i spheroids en kritisk utfordring for langsiktig kultur kreves for å opprettholde og utvikle sine funksjoner som protein uttrykk og morphogenesis. Protokollen presenterer en ny metode for å integrere et perfusable vaskulære nettverk i formet i en microfluidic enhet. For å indusere en perfusable vaskulære nettverket i spheroid, ble angiogenic spirer koblet til microchannels guidet av formet ved å benytte angiogenic faktorer fra menneskelige lunge fibroblaster kultivert i formet. Angiogenic spirer nådde spheroid, sammen med endotelceller co kultivert i spheroid, og dannet et kontinuerlig vaskulære nettverk. Vaskulære nettverket kan perfuse interiøret spheroid uten noen lekkasje. Konstruert vaskulære nettverket kan brukes som en rute ytterligere tilførsel av næringsstoffer og fjerning av avfallsprodukter, etterligne blod sirkulasjon i vivo. Metoden gir en ny plattform i spheroid kultur mot bedre recapitulation av levende vev.

Figur 1 viser en design og bilde av den microfluidic enheten. Den har tre parallelle kanalene, i hvilken kanal 2 inneholder spheroid godt. Kanaler 1 og 3 brukes for HUVEC kultur og kanal 2 er for formet. Hver kanal er separert trapesformet microposts for mønster PDMS. Microposts forhindre hydrogel i kanal 2 lekker inn i 1 og 3 av overflatespenning og tillate utveksling av stoffer mellom formet og HUVECs i microchannels28.
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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 nettverk med en vev modell i en Microfluidic enhet

Protokollen beskriver hvordan å en perfusable vaskulære nettverket i en spheroid. Den formet rundt microenvironment er utviklet for å indusere angiogenese og koble formet til microchannels i en microfluidic enhet. Den tillater perfusjon av spheroid, som er en etterlengtet teknikk i tredimensjonale 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 ...

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