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Bio-ingénierie

Trækkraft mikroskopi integreret med Microfluidics til kemo-aktisk kollektiv migration

Published: October 13, 2019
doi:
10.3791/60415
, , , , ,
1Department of Biomedical Sciences,Korea University, 2Department of Mechanical Engineering,Korea Advanced Institute of Science and Technology, 3Department of Environmental Health,Harvard T.H. Chan School of Public Health

Summary

Kollektiv celle migration i udvikling, sårheling, og kræft metastaser er ofte styret af gradienter af vækstfaktorer eller signalering molekyler. Beskrevet her er et eksperimentelt system, der kombinerer trækkraft mikroskopi med et mikrofluidisk-system og en demonstration af, hvordan man kvantificerer mekanik af kollektiv migration under biokemisk gradient.

Abstract

Celler ændre migrationsmønstre som reaktion på kemiske stimuli, herunder gradienter af stimuli. Cellulære migration i retning af en kemisk gradient, kendt som chemotaxis, spiller en vigtig rolle i udviklingen, immunrespons, sårheling, og kræft metastaser. Mens chemotaxis modulerer migrationen af enkeltceller samt samlinger af celler in vivo, in vitro forskning fokuserer på enkelt-celle chemotaxis, dels på grund af manglen på de rette eksperimentelle værktøjer. For at udfylde dette hul, beskrevet her er et unikt eksperimentelt system, der kombinerer mikrofluidics og micropatterning for at demonstrere virkningerne af kemiske gradienter på kollektiv celle migration. Desuden er trækkraft mikroskopi og enkeltlags stress mikroskopi indarbejdet i systemet for at karakterisere ændringer i cellulær kraft på substratet såvel som mellem tilstødende celler. Som proof-of-koncept, migrering af mikromønstrede cirkulære øer af Madin-Darby hunde nyre (mdck) celler testes under en gradient af hepatocyt vækstfaktor (hgf), en kendt spredning faktor. Det er konstateret, at celler placeret nær den højere koncentration af HGF migrere hurtigere end dem på den modsatte side i en celle ø. Inden for samme ø, cellulære trækkraft er ens på begge sider, men intercellulære stress er meget lavere på siden af højere HGF koncentration. Dette nye eksperimentelle system kan give nye muligheder for at studere mekanik af kemo aktisk migration af cellulære kollektiver.

Introduction

Cellulær migration i biologiske systemer er et fundamentalt fænomen involveret i vævs dannelse, immunrespons og sårheling1,2,3. Cellulære migration er også en vigtig proces i nogle sygdomme som kræft4. Celler migrerer ofte som en gruppe i stedet for enkeltvis, hvilket kaldes kollektiv celle overførsel4,5. For at cellerne skal bevæge sig kollektivt, er det vigtigt at registrere mikromiljøet6. For eksempel, celler opfatter fysisk-kemiske stimuli og reagere ved at ændre motilitet, celle-substrat interaktioner, og celle-celle interaktioner, hvilket resulterer i retningsbestemt migration langs en kemisk gradient7,8, 9,10. Baseret på denne forbindelse, hurtige fremskridt er blevet gjort i Lab-on-a-chip-teknologier, der kan skabe velkontrollerede kemiske mikromiljøer såsom gradient af en chemoattraktant11,12,13 . Mens disse Lab-on-a-chip-baserede mikrofluidics tidligere er blevet brugt til at studere kemo taxaer af cellulære ensemble eller cellulære sfæroider14,15,16,17, de har været anvendt mest i forbindelse med enkelt celle migration18,19,20,21. De mekanismer, der ligger til grund for en cellulær kollektiv respons på en kemisk gradient, er stadig ikke velforstået14,22,23,24,25,26 . Således udvikling af en platform, der muliggør spatiotemporale kontrol af opløselige faktorer samt in situ observation af cellernes biofysiske vil bidrage til at opklare mekanismerne bag kollektiv celle migration.

Udviklet og beskrevet her er en multi-kanaliseret mikrofluidisk system, der muliggør generering af en koncentration gradient af opløselige faktorer, der modulerer migration af mønstrede celle klynger. I denne undersøgelse, hepatocyt vækstfaktor (HGF) er valgt til at regulere migrationen opførsel af Madin-Darby canine nyre (MDCK) celler. Hgf er kendt for at dæmpe celle celle integritet og øge motilitet af cellerne27,28. I det mikrofluidisk system, Fourier omdanne trækkraft mikroskopi og enkeltlags stress mikroskopi er også indarbejdet, som giver mulighed for analyse af motilitet, kontraktile kraft, og intercellulære spændinger induceret af konstituerende celler som reaktion på en hgf Gradient. Inden for samme ø, celler placeret nær den højere koncentration af HGF migrere hurtigere og vise lavere intercellulære stress niveauer end dem på siden med lavere HGF koncentration. Resultaterne tyder på, at dette nye eksperimentelle system er egnet til at undersøge andre spørgsmål på områder, der involverer kollektiv cellulær migration under kemiske gradienter af forskellige opløselige faktorer.

Protocol

Bemærk: litografi af SU-8 forme til stencils (tykkelse = 250 μm) og Micro Channel dele (tykkelse = 150 μm), glas ætsning (dybde = 100 μm), og støbt fabrikation blev udliciteret ved at sende designs ved hjælp computer-aided design software til fabrikanter. 1. fabrikation af Polydimethylsiloxan (PDMS) stencil og mikrokanal Design mikromønsteret af stencil og mikrokanal. Fabrikere eller outsource Su-8 forme (tykkelse på ~ 250 μm for stencils og ~ 150 μm for mikrokan…

Representative Results

For at undersøge kollektiv migration under en kemisk gradient blev et mikrofluidisk system integreret med trækkraft mikroskopi (figur 1). For at opbygge det integrerede system blev polyacrylamid (PA) gel støbt på Special skåret glas, og MDCK-celler blev seedet i mikromønstrede øer lavet af en PDMS stencil. Til dette eksperiment blev der oprettet tolv øer af MDCK-celler (fire rækker med tre kolonner, diameter på ~ 700 μm). Efter celler knyttet til PA gels, PDMS stencil blev fjernet…

Discussion

Kollektiv migration af konstituerende celler er en vigtig proces under udvikling og regenerering, og den migrerende retning styres ofte af den kemiske gradient af vækstfaktorerne4,23. Under kollektiv migration, celler holde interagere med tilstødende celler og underliggende substrater. Sådanne mekaniske interaktioner giver anledning til emergent fænomener som durotaxis42, plithotaxis33og kenotaxis<sup class="xre…

Divulgations

The authors have nothing to disclose.

Acknowledgements

Dette arbejde blev støttet af Koreas National Research Foundation (NRF) tilskud finansieret af den koreanske regering (MSIP) (nr. NRF-2017R1E1A1A01075103), Korea University Grant og BK 21 Plus-programmet. Det blev også støttet af National Institutes of Health (U01CA202123, PO1HL120839, T32HL007118, R01EY019696).

Materials

0.25% trypsin-EDTA (1X) Gibco 25200-056
1 M HEPES buffer solution Gibco 15630-056
1 mm Biopsy punch Integra Miltex 33-31AA-P/25
100 mm petri dishes SPL 10100 100 mm diameter, 15 mm height
14 mm hollow punch ILJIN 124-0571
18 mm Ø Coverslip Marienfeld-Superior 111580 Circular 18 mm, thickness No. 1 (0.13 to 0.16 mm)
2% bis-acrylamide solution Bio-Rad 1610142 Wear protective gloves, clothing, and eye protection.
3-(Trimethoxysilyl)propyl methacrylate (TMSPMA) Sigma-Aldrich 440159-500ML
3-way stopcock Hyupsung HS-T-61N CAUTION: do not use if previously opened. do not resterlize or resuse
30 cm minimum volume line (for pediatric) Hyupsung HS-MV-30 CAUTION: do not use if previously opened. do not resterlize or resuse
35 mm cell culture dish Corning 430165
40% Acrylamide Solution Bio-Rad 1610140 Wear protective gloves, clothing, and eye protection.
75 cm minimum volume line (for pediatric) Hyupsung HS-MV-75 CAUTION: do not use if previously opened. do not resterlize or resuse
acetic acid J.T. Baker JT9508-03
Ammonium persulfate (APS) Bio-Rad 1610700
Antibiotic-Antimycotic Gibco 15240-062
Bottom glass chip MicroFIT 24 x 24 x 1 mm, custom-made, rectangular groove (6 x 12 mm, depth : 100 μm)
Collagen typeI, Rat tail Corning 354236
Custom glass holder Han-Gug Mechatronics custom-made
Dulbecco's Modified Eagle's Medium (DMEM) Welgene LM 001-11
Dulbecco's Phosphate Buffered Saline (PBS) Biowest L0615-500 w/o Magnesium, Calcium
Fetal bovine serum (FBS) Gibco 26140-179
FluoSpheres amine-modified microspheres Invitrogen F8764 0.2 µm, yellow-green fluorescent(505/515)
Hepatocyte Growth Factor (HGF) Sigma-Aldrich H1404-5UG recombinant, human
JuLI stage live cell imaging system NanoEnTek In Automated X-Y-Z stage and fluorsent imaging Incubator-compatible (37 °C and 5% CO2)
Madin-Darby Canine Kidney (MDCK) cell type II
Oxygen plasma system Femto Science CUTE-MPR
Pluronic F-127 Sigma-Aldrich P2443-250G
Rhodamine B isothiocyanate–dextran Sigma-Aldrich R9379-100MG 70 kDa, used to estimate spatiotemporal distribution of HGF in the microfluidic channel
Steril hypodermic needle 18 G KOVAX Trim the tip of the needle and bend it 90 degrees for connecting in/out ports with volume line
Sticky tape 3M/Scotch 810D 33 m x 19 mm
SU-8 master molds MicroFIT 4” diameter, custom-made
sulfosuccinimidyl 6-(4’-azido-2’-nitrophenylamino)hexanoate (Sulfo-SANPAH) Thermo Scientific 22589 Store at -20°C. Store protected from moisture and light.
Sylgard 184 Elastomer Kit Dow Corning PDMS
Syringe pump Chemyx Inc. model fusion 720 withdraw fluid
Syringes KOVAX 1, 3, 5, 10, or 50 cc for using inlet reservoir or outlet syringe pump
tetramethylethylenediamine (TEMED) Bio-Rad 1610800 Wear protective gloves, clothing, and eye protection.
Ultraviolet (UV) lamp UVP LLC 95-0248-02 365 nm wavelength
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Tags

Traction MicroscopyMicrofluidicsCollective Cell MigrationBiochemical GradientCellular ForcesPDMSSU-8 MoldGlass Slide SilanizationPolyacrylamide Gel
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Jang, H., Kim, J., Shin, J. H., Fredberg, J. J., Park, C. Y., Park, Y. Traction Microscopy Integrated with Microfluidics for Chemotactic Collective Migration. J. Vis. Exp. (152), e60415, doi:10.3791/60415 (2019).
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