Name: rotating cell culture systems for human cell culture: human trophoblast cells as a model
Uploaded: 2012-01-18T12:00:00
Description: Watch this Scientific Journal Video about Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model at JoVE.com

Dette arbejde blev støttet af det amerikanske National Institutes of Health tilskud NIH / NICHD # HD051998 (til CAM).

1. Kollagen Bead Forberedelse <ol><li> Før læsning EVTs for 3-D-cellekultur, skal en til at forberede Cytodex-3 microcarrier perler: <ol><li> Afvejes en passende mængde Cytodex-3 perler kræves til eksperimentet. Denne protokol er tilpasset til de 10ml RCCS fartøj, hvor 0.05g af perler er nødvendige. For en 50ml RCCS fartøj, skala i overensstemmelse hermed. I en 50mL autoklaverbar konisk rør, 250 mg Cytodex-3 perler blandes med 12ml Dulbecco fosfatbuffer opløsning (DPBS). Dette beløb er tilstrækkeligt til 5 ...

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Kulturen Teknikken præsenteres her giver efterforskerne med yderst invasive EVT-lignende celler. Det er nu blevet anerkendt, at et tab af differentiering forekommer i monolag på grund af hæmning af cellulære reaktioner på kemiske og molekylære signaler i tre dimensioner (apikale, basale og lateral celleoverfladerne) 10,13. Denne teknik afspejler karakteristika bemærket i livmoderen på invaderende EVT celler. Da proceduren efterligner konventionelle éncellelag vævskultur tid kinetik, men giv...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer </td></tr><tr><td> Cytodex microcarrier perler </td><td> Sigma-Aldrich </td><td> C3275 </td><td></td></tr><tr><td> Roterende cellekultursystem (RCCS) </td><td> Synthecon </td><td> RCCS-D </td><td> Inkluderer rotor base, strømforsyning, 4 disponible RCCS enheder </td></tr><tr><td> RCCS Engangs Enheder </td><td> Synthecon </td><td> Kontakt Synthecon </td><td></td></tr><tr><td> 3 ml Luer-Lock spids sprøjte </td><td> BD </td><td> 309585 </td><td></td></tr><tr><td> 10ml bred-tip serologisk pipette </td><td> BD </td><td> 357504 </td><td></td></tr><tr><td> MEM Alpha </td><td> Invitrogen </td><td> 12561-072 </td><td></td></tr><tr><td> Leibovitz har L-15 medium, pulver </td><td> Invitrogen </td><td> 41300-039 </td><td></td></tr><tr><td> H 2 O, endotoksin gratis </td><td> Fisher </td><td> MT-25-055-CM </td><td></td></tr><tr><td> Natriumbicarbonat </td><td> Sigma-Aldrich </td><td> S-7795 </td><td></td></tr><tr><td> Pepton </td><td> Fisher Scientific </td><td> BP1420-100 </td><td></td></tr><tr><td> Fruktose </td><td> Sigma-Aldrich </td><td> F3510-100 </td><td></td></tr><tr><td> Galactose </td><td> Sigma-Aldrich </td><td> G5388-100 </td><td></td></tr><tr><td> Glucose </td><td> Sigma-Aldrich </td><td> G7528-250 </td><td></td></tr><tr><td> HEPES </td><td> Invitrogen </td><td> 15630-080 </td><td></td></tr><tr><td> L-Glutamin </td><td> Invitrogen </td><td> 25030 </td><td></td></tr><tr><td> Insulin-transferrin-natriumselenid (ITS) </td><td> Sigma-Aldrich </td><td> I1884 </td><td></td></tr><tr><td> FBS </td><td> Invitrogen </td><td> 10437 </td><td></td></tr><tr><td> Penicillin-Streptomycin </td><td> Invitrogen </td><td> 15140 </td><td></td></tr></tbody></table>

rotating cell culture systems for human cell culture: human trophoblast cells as a model

Feltet af menneskelige trofoblasten forskning aids i at forstå den komplekse miljø etableret i løbet placentation. På grund af arten af disse undersøgelser, humane in vivo eksperimenter er umuligt. En kombination af primære kulturer, eksplantation kulturer og trofoblasten cellelinier 1 Støtte vores forståelse af invasion af livmodervæggen 2 og ombygning af uterin spiral arterier 3,4 af extravillous trofoblasten celler (EVTs), som er nødvendig for en vellykket etablering af graviditeten. På trods af den rigdom af viden hentet fra sådanne modeller, er det accepteret, at in vitro-cellekultur modeller ved hjælp af EVT-lignende celle linjers display ændrede cellulære egenskaber i forhold til deres in vivo modstykker 5,6. Cellerne dyrkes i den roterende cellekultur-system (RCCS) display morfologiske, fænotypisk, og funktionelle egenskaber af EVT-lignende celle linjer, der i højere grad efterligner differentiere i uTero EVTs, med øget ekspression af gener medierende invasion (f.eks matrixmetalloproteinaser (MMPs)) og trofoblasten differentiering 7,8,9. Saint Georges Hospital Placental cellelinie-4 (SGHPL-4) (venligst doneret af Dr. Guy Whitley og Dr. Judith Cartwright) er en EVT-lignende cellelinie, der blev brugt til test i RCCS. Udformningen af ​​RCCS kultur skib er baseret på princippet om, at organer og væv fungerer i en tre-dimensionel (3-D) miljø. På grund af de dynamiske dyrkningsbetingelser i skibet, herunder betingelser for fysiologisk relevante shear, celler dyrket i tre dimensioner danner aggregater baseret på naturlige cellulære tilhørsforhold og differentiere sig til organotypiske væv-agtige forsamlinger 10,11,12. Opretholdelse af en væske bane giver en lav-forskydning, lav turbulens miljø, der svarer til tilstande, der findes in vivo. Sedimentation af dyrkede celler er imødegås ved at justere rotationenhastighed RCCS at sikre en konstant frit fald af celler. Gasbørs sker gennem et permeabelt hydrofob membran placeret på bagsiden af ​​bioreaktor. Ligesom deres forældre væv in vivo, er RCCS-dyrket celler i stand til at reagere på kemiske og molekylær gradienter i tre dimensioner (dvs. på deres apikale, basale og laterale overflader), fordi de dyrkes på overfladen af porøse microcarrier perler. Når der dyrkes som to-dimensionelle monolag på befæstede arealer som plastik, er cellerne berøvet denne vigtige kommunikation på deres basale overflade. Derfor, den rumlige begrænsninger, som miljøet dybt indflydelse på, hvordan celler fornuft og afkode signaler fra det omgivende mikromiljø, hvilket antyder en vigtig rolle for de 3-D miljø 13. Vi har brugt RCCS til ingeniør biologisk meningsfuld 3-D modeller af forskellige menneskelige epitelvæv 7,14,15,16. Mange tidligere rapporter har DEMonstrated at cellerne dyrkes i RCCS kan påtage sig fysiologisk relevante fænotyper, der ikke har været muligt med andre modeller 10,17-21. Sammenfattende repræsenterer kulturen i RCCS en nem, reproducerbar, high-throughput platform, der giver et stort antal af differentierede celler, der gøres til genstand for en bred vifte af eksperimentelle manipulationer. I det følgende protokol, ved hjælp EVTs som et eksempel vi klart beskrive de nødvendige skridt til tre-dimensionelt kultur klæbende celler i RCCS.

Watch this Scientific Journal Video about Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model at JoVE.com

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

Traditionel, todimensionale cellekultur teknikker ofte resultere i ændrede egenskaber med hensyn til differentiering markører, cytokiner og vækstfaktorer. Tre-dimensionel cellekultur i den roterende cellekultur-system (RCCS) genopretter udtryk for mange af disse faktorer, som vist her med en extravillous trofoblasten cellelinje.

Roterende Cell Culture Systems for Human Cell Culture: Human trofoblasten celler som en model

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

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