Name: concentric gel system to study the biophysical role of matrix microenvironment on 3d cell migration
Uploaded: 2015-04-03T15:00:00
Description: Watch this Scientific Journal Video about Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration at JoVE.com

Forfatterne takker W. Sun og K. Jansen for de kritiske diskussioner, og anerkende støtte fra Nano Biomekanik Lab på National University of Singapore. NAK anerkender støtte fra et Marie Curie IIF Fellowship.

1. Cell Høst <ol><li> Opnå MD-MBA-231-celler fra 37 ° C, 5% CO 2-inkubator. Frigør celler fra vævskulturplade anvendelse af 0,5% trypsin-EDTA-opløsning. Brug 1 ml trypsin-EDTA-opløsning for celle dyrket i en T25 kolbe. </li><li> Pellet-celler i et 15 ml konisk rør ved centrifugering ved 200 x g i 4 min, aspireres supernatanten og resuspender cellerne i 5 ml dyrkningsmedium. </li><li> Tæl celletæthed, ρ, anvendelse af et hæmocytometer. Bemærk: For at forberede celle-seedede indre...

<ol>
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In this protocol we describe an in vitro assay to study the 3D migrational behavior of cells in matrix environments that topologically resemble ECMs encountered in vivo. There are three main strengths of this assay as compared to other currently available methods. First, this assay allows one to simultaneously examine the cell migration mechanisms at both population level and individual cell level. This opens up possibilities of studying collective cell migration13, which has to date been lar...

Migration af celler spiller en central rolle i forskellige fysiologiske reaktioner, såsom fosterudvikling, hæmostase, og immunrespons samt i patologiske processer, såsom vaskulære sygdomme, inflammation og cancer 1. Dissekere de biokemiske og biofysiske faktorer bag celle migration er derfor fundamentalt vigtigt ikke kun at forstå de grundlæggende principper for cellulære funktioner, men også at fremme forskellige biomedicinske applikationer, såsom i tissue engineering, anti-metastase og anti-inflammatorisk lægemiddeludvikling. Da in vivo observation er teknisk udfordrende, har en stor indsats været fokuseret på in vitro sammenfatning af celle migration. In vitro-metoder til at studere celle migration er stort set udviklet til analyser på todimensionale (2D) overflader, især den bunden eller sårheling assay 2. Sådanne analyser giver simpel forsøgsopstilling, let hus-cell imaging, og give nyttig indsigt i forskellige biokemiske mekanismer bag celle migration. Men disse assays ikke højde for ekstracellulær matrix (ECM) arkitektur og remodeling, som er kritiske aspekter i forståelse in vivo migration. For nylig er det blevet stadig mere klart, at en 3D-kultur-model, ofte i collagen-baserede matricer 3, tilvejebringer en platform, der bedre ligner in vivo-situationen. Faktisk celler udviser migrational dynamik, der er forskellige fra dem på 2D overflader, især på grund af de forskellige dimensioner af miljøet 4. Desuden de biofysiske og mekaniske egenskaber af matrixen følsomt påvirke cellemigration 5, herunder i forbindelse med tumorcelleinvasion 6. Her præsenterer vi en metode til at studere 3D celle migration adfærd i ECM med biofysiske egenskaber, der kan nemt varieres med forberedelse betingelser. Cellernepodet i en &quot;indre gel&quot; og får lov til at slippe ind og invadere den oprindeligt acellulær &quot;ydre gel&quot;. Metoden bygger på cellens evne til at genkende tilstedeværelsen af, og tilbøjelighed til at vandre ind, cellefrie regioner i den ydre gel, som er tæt knyttet til celle mechanosensing 7. I denne undersøgelse beskæftiger vi kollagen netværk som de ECM&#39;er invaderet af meget invasive brystkræftceller, MD-MBA-231. De mekaniske egenskaber og mikrostruktur af både de indre og ydre geler kan indstilles 8 og kendetegnet 9 for at opnå fysiologisk relevante betingelser. Genopbygning og analyse af celle spor tillader detaljerede kvantitative undersøgelse af spatiotemporale migration adfærd på både befolkningen og enkelte celle niveau. Vigtigt er det, at opsætning af koncentriske gelsystem efterligner in vivo væv topologi ved at migrere celler, især invaderer kræftceller, hvilket giver vigtig indsigt i konfronteretde fysiske mekanismer cellevandring og metastase.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Cell culture incubator</td>
			<td>Fisher Scientific Pte Ltd</td>
			<td>Model: 371, S/No 318854-6055</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Nikon A1R</td>
			<td></td>
			<td>Inverted confocal laser scanning microscope equipped with incubator chamber</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td>Life Technologies</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Life Technologies</td>
			<td>10082-147</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent CellTracker dye CMTMR</td>
			<td>Life Technologies</td>
			<td>C2927</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass-bottom dish</td>
			<td>IWAKI Cell Biology</td>
			<td>3931-035</td>
			<td>35 mm diameter dish with 12 mm diameter glass-bottom well</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>iN CYTO</td>
			<td>DHC-N01 (Neubauer Improved)</td>
			<td></td>
		</tr>
		<tr>
			<td>Microprocessor pH meter</td>
			<td>Hanna Instruments</td>
			<td>pH 211</td>
			<td></td>
		</tr>
		<tr>
			<td>Nutragen Collagen</td>
			<td>Advanced BioMatrix</td>
			<td>#5010-D</td>
			<td>Acid-solubilized bovine collagen type I (stock pH ~ 2)</td>
		</tr>
		<tr>
			<td>Objective lens</td>
			<td>Nikon</td>
			<td></td>
			<td>CFI Super Plan Fluor ELWD ADM 20XC, W.D. 8.2-6.9mm, NA 0.45.</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Sartorius</td>
			<td>S/No 29153352</td>
			<td>Basic pH Meter PB-11</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Life Technologies</td>
			<td>15400-054</td>
			<td></td>
		</tr>
	</tbody>
</table>

concentric gel system to study the biophysical role of matrix microenvironment on 3d cell migration

Cellernes evne til at migrere er afgørende i en lang række cellefunktioner hele livet fra fosterudvikling og sårheling til tumor og cancer metastaser. Trods intens forskningsindsats, de grundlæggende biokemiske og biofysiske principper cellemigration stadig ikke fuldt forstået, især i de fysiologisk relevante tredimensionale (3D) mikromiljøer. Her beskriver vi et in vitro assay designet til at tillade kvantitativ undersøgelse af 3D cellemigration adfærd. Metoden udnytter cellens mechanosensing evne og tilbøjelighed til at vandre ind tidligere ubesatte ekstracellulære matrix (ECM). Vi bruger invasion af meget invasive brystcancerceller, MDA-MB-231, i kollagengeler som et modelsystem. Kan overvåges Udbredelsen af ​​cellepopulationen og migrationen dynamik individuelle celler i løbet ugers dyrkning ved hjælp af live-cell imaging og analyseres for at udtrække spatiotemporally-løst data. EndvidereEr fremgangsmåden let at tilpasse til forskellige ekstracellulære matricer, hvilket giver en enkel, men effektiv måde at undersøge den rolle, biofysiske faktorer i mikromiljø på celle migration.

Den koncentriske gel assay præsenteres her blev udført under anvendelse af meget invasive brystcancerceller, MDA-MB-231, med 2,4 mg / ml indre kollagengel og cellepodning densitet = 2 x 10 6 celler / ml, som et eksempel. Som vist i figur 2, typisk efter et par dages dyrkning blev cellerne tilsidesat indre ydre gel interface og begyndte at invadere ydre gel. Cellepopulationen spredes overvejende radialt udad. Polymerisationsbetingelserne af den ydre gel kan modif...

Watch this Scientific Journal Video about Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration at JoVE.com

Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration

De mekaniske egenskaber og mikrostruktur af den ekstracellulære matrix påvirker kraftigt 3D migration af celler. En in vitro metode til at studere spatiotemporale cellemigrering adfærd i biofysisk variable miljøer på både befolkning og individuelle celle niveauer, er beskrevet.

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