JoVE Journal > Bioengineering
Summary
Abstract
Introduction
Protocol
Risultati
Discussion
Divulgazioni
Acknowledgements
Materials
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Bioingegneria

Procedure for udvikling af multi-dybde cirkulære Tværsnit Endothelialized Microchannels-on-a-chip

Published: October 21, 2013
doi:
10.3791/50771
, , ,
1Lane Department of Computer Science and Electrical Engineering,West Virginia University, 2Department of Cell Biology and Neuroscience,University of California at Riverside

Summary

En microchannels-on-a-chip platform blev udviklet af en kombination af fotolitografisk reflowable photoresist teknik, blød litografi og mikrofluidik. Den endothelialized microchannels platform efterligner den tredimensionelle (3D) geometri in vivo mikrokar kører under kontrollerede kontinuerlig perfusion flow, giver mulighed for høj kvalitet og real-time scanning, og kan anvendes til microvascular forskning.

Abstract

Indsatsen har været fokuseret på at udvikle in vitro assays til studiet af mikrokar fordi in vivo dyreforsøg er mere tidskrævende, dyrt og observation og kvantificering er meget udfordrende. Imidlertid konventionel in vitro mikrokar assays har begrænsninger, når det repræsenterer in vivo mikrokar med hensyn til tredimensionale (3D) geometri og tilvejebringe kontinuerlig fluidstrømning. Ved hjælp af en kombination af fotolitografisk reflowable photoresist teknik, blød litografi og MicroFluidics, har vi udviklet en multi-dybde cirkulære tværsnit endothelialized microchannels-on-a-chip, som efterligner den 3D-geometri in vivo mikrokar og kører under kontrollerede kontinuerlig perfusion flow. En positiv reflowable fotoresist blev anvendt til at fremstille en master støbeform med et halvcirkelformet tværsnit mikrokanalplade netværk. Ved tilpasning og limning af de to polydimetylsiloxan (PDMS) microchannels REPLlemstore fra master formen blev en cylindrisk mikrokanalplade netværk oprettet. De diametre microchannels kan være velkontrolleret. Desuden viste primære humane navlevene endotelceller (HUVEC'er) podet inde i chippen, at cellerne foret den indvendige overflade af mikrokanaler under kontrollerede perfusion varig i et tidsrum mellem 4 dage til 2 uger.

Introduction

Mikrokar, som en del af cirkulationssystemet, medierer samspillet mellem blod og væv støtte metaboliske aktiviteter, definere væv mikromiljø, og spiller en afgørende rolle i mange sundheds-og patologiske tilstande. Sammenfatning af funktionelle mikrokar in vitro kunne give en platform for studiet af komplekse vaskulære fænomener. Imidlertid konventionel in vitro mikrokar assays, såsom endotelcellemigration assays, endotelceller rørdannelse assays og rotter og mus aortaringen assays, er i stand til at genskabe in vivo mikrokar med hensyn til tredimensionale (3D) geometri og kontinuerlig strøm kontrol 1-8. Undersøgelser af mikrokar anvendelse af dyremodeller og in vivo assays, såsom hornhinde angiogenese assay, kyllingechorioallantoinmembranen membran angiogenese assay, og Matrigel plug assay, er mere tidskrævende, høj i pris, udfordrende med hensyn til observation og kvantificeringer ogrejser etiske spørgsmål 1, 9-13.

Fremskridt i micromanufacturing og mikrofluid chip teknologier har muliggjort en bred vifte af indsigt i biomedicinske videnskaber, mens begrænse de høje eksperimentelle omkostninger og kompleksiteter i forbindelse med dyr og in vivo studier 14, såsom let og stramt kontrollerede biologiske forhold og dynamiske fluidisk miljøer, hvilket ikke ville have været muligt med konventionelle Makroskalaplacering teknikker.

Her præsenterer vi en tilgang til at konstruere en endothelialized microchannels-on-a-chip, som efterligner den 3D-geometri in vivo mikrokar og kører under kontrollerede kontinuerlig perfusion flow ved hjælp af en kombination af fotolitografisk reflowable photoresist teknik, blød litografi og mikrofluidik.

Protocol

1.. Fotolitografi Fabrikation af Fotoresist Master Mold Følgende protokol viser processen for at fremstille de mikrokanaler med diametre på mellem 30-60 um. At få en mikrokanalplade med en mindre diameter (mindre end 30 um), en enkelt spin-coating af fotoresist er nødvendig. Overfør reflow fotoresist fra køleskabet ved 4 ° C til renrummet 24 hr før brug og lad den varme op til stuetemperatur. Rens en siliciumskive og bage det i en time ved 150 ° C for at tillade…

Representative Results

Vores tilgang til at fremstille multi-dybde mikrokanalplade netværk efterligner komplekse 3D geometrier af in vivo mikrokar, hvor mikrokanalerne har afrundede tværsnit 15.. Derudover diametrene af moder forgrenende kanaler og datter kanaler omtrent adlyde Murray lov til opretholdelse af fluidumstrømmen på et krævede niveau, så den samlede kanal modstanden er lav og strømningshastigheder er mere ensartet i hele netværket 16-18. De processer og resultater for fremstilling af en halvc…

Discussion

1.. Master skimmel fabrikation

En af de designe og vejledende principper for vaskulær morfometri er kendt som Murray lov 16, hvor det hedder, at fordelingen af fartøjets diametre hele nettet reguleres af minimum energi overvejelse. Det fastslås også, at terningen af ​​diametre på en forælder fartøj på en tvedeling er lig summen af ​​de terninger af diametre datter fartøjer ( <img alt="Ligning 1" fo:content-width="0.9in" fo:src="/files/ftp_upload/50771/50771eq1….

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

Denne forskning blev delvist støttet af National Science Foundation (NSF 1.227.359), WVU EPSCoR program finansieret af National Science Foundation (EPS-1.003.907), WVU ADVANCE kontor sponsoreret af National Science Foundation (1.007.978) og WVU PSCoR hhv. Den microfabrication arbejde blev udført i WVU fælles forskning Faciliteter (renrumsfaciliteter) samt Mikrofluid Integrativ Cellular Forskning i Chip Laboratory (mikrochip Lab) ved West Virginia University. Den konfokal imaging blev udført på WVU Microscope Imaging faciliteten.

Materials

Reagent/Material
Reflow Photoresist AZ Electronic Materials AZP4620
Developer AZ Electronic Materials AZ 400K
PDMS Dow Corning Corporation Sylgard 184
MCDB 131 Culture Medium Invitrogen 10372-019
NacBlue Nuclei Staining Invitrogen H1399
PKH Red Stain Sigma MINI26 and PKH26GL
Fibronectin Gibco PHE0023
L-Glutamine Sigma G7513
Phosphate Buffered Saline Invitrogen 14040-133
HEPES Buffered Saline Solution Lonza CC-5024
Trypsin/EDTA Invitrogen 25300-062
Trypsin Neutralizing Solution Lonza CC-5002
PDMS Curing Agent Dow Corning Corporation Sylgard 184
Primary Human Umbilical Vein Endothelial Cells Lonza CC-2517
Fetal Bovine Serum Lonza 14-501F
Diluent C Sigma CGLDIL
Hoechst33342 Invitrogen, Molecular Probes R37605
Dextran Sigma 95771
3.5% Paraformaldehyde Electron Microscopy Science 15710-S
Equipment
Spinner Laurell Technologies Corporation WS-400BZ-6NPP/LITE
Desiccator BelArt Products 999320237
Inverted Microscope Nikon Eclipse Ti
Syringe Pump System Harvard Apparatus PHD Ultra
Laminar Biosafety Hood Thermo Scientific 1300 Series A2
Planetary Centrifugal Mixer Thinky ARE-310
Isotemp Oven Fisher Scientific 13-246-516GAQ
Optical Microscope Zeiss Invertoskop 40C
Plasma Cleaner Harrick Plasma PDC-32G
Hotplate Barnstead/Thermolyne Cimarec SP131635
Laser Scanning Confocal Microscope Zeiss LSM 510
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Tags

Multi-depth Circular Cross-sectional MicrochannelsEndothelialized MicrochannelsIn Vitro Microvessels3D GeometryContinuous Fluid FlowPhotolithographic ReflowSoft LithographyMicrofluidicsPDMSPrimary Human Umbilical Vein Endothelial Cells (HUVECs)Perfusion
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Li, X., Mearns, S. M., Martins-Green, M., Liu, Y. Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip. J. Vis. Exp. (80), e50771, doi:10.3791/50771 (2013).
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