Name: three-dimensional cell culture model for measuring the effects of interstitial fluid flow on tumor cell invasion
Uploaded: 2012-07-25T12:00:00
Description: Watch this Scientific Journal Video about Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion at JoVE.com

1. Analys Set-up <ol><li> Tina en liten alikvot (&lt;500 | il) av Matrigel på is vid 4 ° C (ca 2 h). </li><li> Förbered gel recept (se t.ex. volymer i tabellen nedan): 10x PBS (1x totalt volym), 1 N natriumhydroxid (motsvarande 0,023 volymer till kollagen, eller per kollagen tillverkarens rekommendationer, i förekommande fall), skriver Matrigel och kollagen jag slutkoncentrationer av 1 mg / ml och 1,3 mg / ml (andra matrisformuleringar kan användas beroende på celltyp och experimentet). </li></ol><p class="jove_...

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Här har vi beskrivit en metod för att kvantifiera effekten av interstitiell flöde på tumörcellsinvasion, med användning av celler som är inbäddade i en 3-D-matris i en cellodling insatsen. Denna och liknande metoder har använts för att studera effekten av interstitiell flöde på ett antal olika celltyper 13-15, 17. Vårt tillvägagångssätt härmar delvis matrisen mikromiljö av tumören med hjälp av typ I kollagen och Matrigel som innehåller proteiner som finns i källaren membran epitelial vä...

<table border="1"><tbody><tr><td> Namnet på reagens </td><td> Företaget </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> Kollagen (Råtta svans) </td><td> BD </td><td> 354.236 </td><td> Hålla steril </td></tr><tr><td> Millicell cellkultur insatsen </td><td> Millipore </td><td> PI8P01250 </td><td> 8 | am pordiameter, polykarbonatmembran </td></tr><tr><td> Matrigel </td><td> BD </td><td> 354.234 </td><td> Hålla steril </td></tr><tr><td> PBS </td><td> Sigma Aldrich </td><td> 100M-8202 </td><td> 10x för att förbereda gellösningen, 1x för tvättningssteg </td></tr><tr><td> Natriumhydroxid, 1,0 N lösning </td><td> Sigma Aldrich </td><td> S2770 </td><td> Hålla steril </td></tr><tr><td> DMEM 1X </td><td> Cellgro </td><td> 10-013-CV </td><td> Hålla steril </td></tr><tr><td> Fetalt bovint serum </td><td> Atlanta Biologicals </td><td> 511.150 </td><td> Hålla steril </td></tr><tr><td> Penicillin streptomycin </td><td> Cellgro </td><td> 30002CI </td><td> Hålla steril </td></tr><tr><td> Triton X-100 </td><td> Sigma Aldrich </td><td> X100-500 ml </td><td> 0,5% i PBS </td></tr><tr><td> Paraformaldehyd </td><td> Fisher Scientific </td><td> 04042-500 </td><td> 4% i PBS </td></tr><tr><td> Avjoniserat vatten </td><td></td><td></td><td> Hålla steril </td></tr><tr><td> 4 &#39;,6-diaminido-2-fenylindol (DAPI) </td><td> MP Biomedicals </td><td> 0215757401 </td><td> 1 mg / ml stamlösning </td></tr><tr><td> Monteringslösning </td><td> Thermo Scientific </td><td> TA-030-FM </td><td></td></tr><tr><td> Trypsin-EDTA </td><td> Cellgro </td><td> 25-052-Cl </td><td> Hålla steril </td></tr></tbody> &lt;/ Table&gt;

three-dimensional cell culture model for measuring the effects of interstitial fluid flow on tumor cell invasion

Tillväxten och utvecklingen av de flesta solida tumörer är beroende på den initiala omvandlingen av cancerceller och deras svar på stroma-associerad signalering i tumören mikromiljö 1. Tidigare har forskning om tumören mikromiljö främst inriktad på tumör-stroma interaktion 1-2. Innehåller dock tumören mikromiljö också en mängd biofysiska krafter, vars effekter fortfarande dåligt kända. Dessa krafter är biomekaniska konsekvenser av tumörtillväxt som leder till förändringar i genuttryck, celldelning, differentiering och invasion 3. Matrix täthet 4, 5-6 styvhet och struktur 6-7, interstitialvätska tryck 8, och interstitiell vätskeflöde 8 är alla förändras under cancer progression. Interstitial vätska strömmar i synnerhet är högre i tumörer jämfört med normala vävnader 8-10. Den beräknade interstitiell vätska flow hastigheter uppmättes och befanns vara i intervallet 0,1-3 | im s -1, beroende på tumörstorlek och differentiering 9, 11. Detta beror på förhöjd interstitiellt vätsketryck som orsakas av tumör-inducerad angiogenes och ökad vaskulär permeabilitet 12. Interstitial vätska strömma har visat sig öka invasion av cancerceller 13-14, vaskulära fibroblaster och glatta muskelceller 15. Denna invasion kan bero på autologa kemotaktiska gradienter skapas runt cellerna i 3-D-16 eller ökas matrix-metalloproteinas (MMP)-expression 15, kemokin sekretion och celladhesionsmolekyl uttryck 17. Emellertid är den mekanism genom vilken celler avkänna vätskeflödet inte väl förstådda. Förutom att förändra tumören cellbeteende, modulerar interstitiell fluidflöde aktiviteten hos andra celler i tumören mikromiljö. Det är förenat med (a) körning differentiering av fibroblaster i tumör-främjande myofibroblaster 18, (b) transport av antigener och andra lösliga faktorer till lymfkörtlar 19, och (c) modulering av lymfatiska endotelceller morfogenes 20. Tekniken som presenteras här innebär interstitialvätska flöde på celler in vitro och kvantifierar dess effekter på invasion (figur 1). Denna metod har publicerats i flera studier för att mäta effekterna av vätskeflöde för stromal och cancer cellinvasion 13-15, 17. Genom att ändra matriskompositionen, celltyp och cellkoncentrationen, kan denna metod tillämpas på andra sjukdomar och fysiologiska system för att studera effekterna av interstitiell flöde på cellulära processer såsom invasion, differentiering, proliferation och genexpression.

Watch this Scientific Journal Video about Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion at JoVE.com

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

Interstitial vätska flöde är förhöjd i fasta tumörer och kan modulera tumörcellsinvasion. Här beskriver vi en metod att applicera interstitiell fluidflöde till celler inbäddade i en matris och sedan mäta dess effekt på cellinvasion. Denna teknik kan lätt anpassas för att studera andra system.

Tredimensionell cellkulturmodell för mätning av effekterna av interstitialvätska Flow på Tumörcellinvasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

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