Name: isolation of mammary epithelial cells from three-dimensional mixed-cell spheroid co-culture
Uploaded: 2012-04-30T12:00:00
Description: Watch this Scientific Journal Video about Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture at JoVE.com

1. Etablering af tre-dimensionelle cokulturer <ol><li> Dagen før indførelse af co-kulturer, fjernes Matrigel fra fryseren og tø på is i en 4 ° C køleskab natten over. </li><li> På dagen for eksperimentet forberede medium til co-kultur, som svarer til medium for epitelceller, der skal anvendes (HMEC medium i dette eksempel: DME/F12 medium med 10% bovint kalveserum, 5 ug / ml insulin , 1 ug / ml hydrocortison og 1 ng / ml EGF). Holde mediet på is i mindst 1 time før blanding med Matrigel. </li><li> At fortynde ...

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Fremgangsmåden præsenteres her repræsenterer en enkel fremgangsmåde til at analysere virkningen af ​​stromacellerne fibroblaster på epitelceller, herunder tumorceller. Det giver mulighed for direkte celle-celle interaktion i et tredimensionalt forbindelse understøttet af ekstracellulær basalmembranmatrix, samtidig med at lette ekstraktion af celler fra matrixen til yderligere analyse. Dette kan anvendes til studiet af tumorassocierede stromata, som fibroblastlinier kan manipuleres til at påvirke signalerings...

<table border="1"><tbody><tr><td> Navn reagens </td><td> Firma </td><td> Katalog nummer </td><td> Comments </td></tr><tr><td> BD Matrigel </td><td> BD Biosciences </td><td> 356237 </td><td> Phenolrødtfrit </td></tr><tr><td> Hyclone Klassisk Flydende Medium: DMEM F12 </td><td> ThermoScientific </td><td> SH30023.01 </td><td></td></tr><tr><td> Hyclone bovint kalveserum </td><td> ThermoScientific </td><td> SH3007303 </td><td></td></tr><tr><td> Insulin </td><td> EMD Kemikalier </td><td> 407709 </td><td> Opløses i 0.005N HCI; 0,22 um filter </td></tr><tr><td> Hydrocortison </td><td> Sigma-Aldrich </td><td> H4001 </td><td> Opløses i 50% EtOH, 0,22 um filter </td></tr><tr><td> EGF </td><td> Sigma-Aldrich </td><td> E9644 </td><td> Opløses i 10 mM eddikesyre, 0,22 um filter </td></tr></tbody></table>

isolation of mammary epithelial cells from three-dimensional mixed-cell spheroid co-culture

Mens enorme indsats er gået ind i at identificere signalveje og molekyler der er involveret i normale og maligne celle adfærd 1-2, meget af dette arbejde er blevet udført ved hjælp af klassiske to-dimensionelle cellekulturmodeller, der giver mulighed for nem celle manipulation. Det er blevet klart, at intracellulære signalveje påvirkes af ekstracellulære kræfter, herunder dimensioner og celle overfladespænding 3-4. Flere tiltag er blevet taget til at udvikle tre-dimensionelle modeller, der mere præcist repræsenterer biologisk væv arkitektur 3. Selv om disse modeller har multi-dimensionalitet og arkitektoniske understreger undersøgelse af de deraf følgende virkninger på celler er mindre bekvem end i to-dimensionelle vævskultur på grund af begrænsninger i de modeller og vanskeligheden ved at udvinde celler til efterfølgende analyse. Den vigtige rolle af mikromiljøet omkring tumorer i tumorigenese og tumor adfærd is stigende grad anerkendt 4. Tumorstroma består af flere celletyper og ekstracellulære molekyler. Under tumorudvikling der er bidirektionale signaler mellem tumorceller og stromaceller 5. Selv om nogle faktorer, der deltager i tumor-stroma CO-EVOLUTION er blevet identificeret, er der stadig et behov for at udvikle enkle teknikker til systematisk at kortlægge og studere den fulde vifte af disse signaler 6. Fibroblaster er den mest rigelige celletype i normale eller tumorassocieret stroma væv, og bidrager til aflejring og vedligeholdelse af basalmembran og parakrine vækstfaktorer 7. Mange grupper er anvendt tredimensionelle dyrkningssystemer at undersøge rollen af fibroblaster på forskellige cellulære funktioner, herunder tumorrespons på terapier, immunceller, signalmolekyler, proliferation, apoptose, angiogenese og invasion 8-15. Vi har optimeret en simpel metode til æselEssing virkninger brystkirtlerne fibroblaster på mammale epitelceller anvendelse af et kommercielt tilgængeligt ekstracellulær matrix model til at skabe tredimensionale kulturer af blandede cellepopulationer (co-kulturer) 16-22. Med fortsat co-dyrkning af cellerne dannelse af sfæroider med fibroblaster klynger i det indre og epitelcellerne i vid udstrækning på det ydre af sfæroider og danne multi-cellulære fremspring i matrixen. Manipulation af fibroblaster, som fører til ændret epitelcelle invasionsevne let kan kvantificeres ved ændringer i antallet og længden af epitel fremskrivninger 23. Endvidere har vi udviklet en fremgangsmåde til isolering af epitelceller ud af tredimensionale co-kultur, der letter analyse af virkningerne af fibroblast eksponering på epitel adfærd. Vi har fundet, at virkningen af ​​co-dyrkning fortsat i flere uger efter epitelcelle isolering, tillader rigelig tid til at udføre multiple assays. Denne fremgangsmåde kan tilpasses til cellervarierende malignt potentiale og kræver ingen specialiseret udstyr. Denne teknik muliggør hurtig evaluering af in vitro-celle-modeller under flere betingelser, og de ​​tilsvarende resultater kan sammenlignes med in vivo dyreforsøg vævsmodeller samt humane vævsprøver.

Watch this Scientific Journal Video about Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture at JoVE.com

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

En simpel fremgangsmåde er beskrevet til at analysere virkningerne af væv-fibroblaster på tilknyttede epitelceller. Kombinationen af ​​denne fremgangsmåde og tredimensionale vævskultur kan lette analyse af celler efter isolering fra 3D. Teknikken kan anvendes til celler af forskellige maligne potentiale, så systematisk undersøgelse af virkningerne af tumorassocierede stromata på tumorceller.

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