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 arbeidet ble støttet av det amerikanske National Institutes of Health stipend NIH / NICHD # HD051998 (til CAM).

1. Kollagen Bead Forberedelse <ol><li> Før lasting EVTs for 3-D cellekultur, må man forberede Cytodex-3 microcarrier perler: <ol><li> Vei opp riktig mengde Cytodex-3 perler kreves for forsøket. Denne protokollen er tilrettelagt for 10ml RCCS fartøy, der 0.05g av perler er nødvendig. For en 50ml RCCS fartøy, skala tilsvarende. I en 50ml autoklaverbar konisk tube, bland 250 mg Cytodex-3 perler med 12ml Dulbecco fosfatbufret løsning (DPBS). Dette beløpet er tilstrekkelig for 5 RCCS fartøy. </li></ol></li><li> S?...

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Kulturen teknikken som presenteres her gir etterforskere med svært invasiv EVT-lignende celler. Det har nå blitt anerkjent som et tap av differensiering skjer i monolayers grunn av hemming av cellulære responser på kjemiske og molekylære signaler i tre dimensjoner (apikale, basal og lateral celleoverflaten) 10,13. Denne teknikken reflekterer kjennetegn bemerket i utero på invaderende EVT celler. Som prosedyren etterligner konvensjonelle monolayer vev kultur tid kinetikk, men gir celler med diff...

<table border="1"><tbody><tr><td> Navn på reagens </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> Cytodex microcarrier perler </td><td> Sigma-Aldrich </td><td> C3275 </td><td></td></tr><tr><td> Roterende Cell Culture System (RCCS) </td><td> Synthecon </td><td> RCCS-D </td><td> Inkluderer rotor base, strømforsyning, 4 disponibel RCCS enheter </td></tr><tr><td> RCCS Engangs Units </td><td> Synthecon </td><td> Kontakt Synthecon </td><td></td></tr><tr><td> 3ml Luer-Lock spiss sprøyte </td><td> BD </td><td> 309585 </td><td></td></tr><tr><td> 10ml wide-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> Natriumbikarbonat </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> Galaktose </td><td> Sigma-Aldrich </td><td> G5388-100 </td><td></td></tr><tr><td> Glukose </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-Natrium selenitt (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 av menneskelige trophoblast forskning hjelpemidler i å forstå det komplekse miljøet etablert i løpet placentation. På grunn av disse studiene, er menneskelig in vivo eksperimentering umulig. En kombinasjon av primær kulturer, eksplantering kulturer og trophoblast cellelinjer som en støtte for vår forståelse av invasjonen av livmorveggen 2 og ombygging av livmor spiral arterier 3,4 av extravillous trophoblast celler (EVTs), som er nødvendig for en vellykket etablering av svangerskapet. Til tross for vell av kunnskap gleaned fra slike modeller, er det akseptert at in vitro cellekultur modeller ved hjelp av EVT-lignende cellelinjene vise endres cellular egenskaper når sammenlignet med sine in vivo kolleger 5,6. Celler dyrket i den roterende cellekultur system (RCCS) display morfologiske, fenotypisk og funksjonelle egenskaper EVT-lignende cellelinjene som nærmere etterligner differensiere i uTero EVTs, med økt uttrykk av gener mediere invasjon (f.eks matrix metalloproteinaser (MMPs)) og trophoblast differensiering 7,8,9. Saint Georges Hospital placenta celle Line-4 (SGHPL-4) (vennlig donert av Dr. Guy Whitley og dr. Judith Cartwright) er en EVT-lignende cellelinje som ble brukt for testing i RCCS. Utformingen av RCCS kultur fartøyet er basert på prinsippet om at organer og vev fungerer i en tredimensjonal (3D) miljø. På grunn av den dynamiske kulturen forholdene i fartøyet, herunder vilkår for fysiologisk relevante skjær, celler dyrket i tre dimensjoner skjemaet tilslag basert på naturlige cellulære tilhørighet og differensieres til organotypic vev-liknende forsamlinger 10,11,12. Opprettholdelsen av et fluid bane gir en lav skjæring, lav turbulens miljø som ligner forholdene funnet in vivo. Sedimentasjon av dyrkede celler er motvirkes ved å justere rotasjonenHastigheten på RCCS å sikre en konstant fritt fall av celler. Gassutveksling skjer gjennom en permeabel hydrofob membran plassert på baksiden av bioreaktor. Som sine foreldre vev in vivo, RCCS dyrket cellene er i stand til å svare på kjemisk og molekylær gradienter i tre dimensjoner (dvs på sin apikale, basal og sideflatene) fordi de er dyrket på overflaten av porøse microcarrier perler. Når vokst som to-dimensjonale monolayers på tette flater som plast, er celler fratatt denne viktige kommunikasjonen på sitt basal overflaten. Følgelig, den romlige begrensninger pålagt av miljøet dypt påvirke hvordan cellene fornuft og dekode signalene fra omkringliggende mikromiljøet, og dermed innebærer en viktig rolle for 3-D miljø 13. Vi har brukt RCCS til ingeniør biologisk meningsfylt 3-D modeller av ulike menneskelige epitelvev 7,14,15,16. Faktisk, mange tidligere rapporter har DEMonstrated at celler dyrket i RCCS kan anta fysiologisk relevante fenotyper som ikke har vært mulig med andre modeller 10,17-21. I sammendraget, representerer kulturen i RCCS en enkel, reproduserbar, high-throughput plattform som gir et stort antall differensierte celler som er mottagelig for en rekke eksperimentelle manipulasjoner. I det følgende protokoll, bruke EVTs som et eksempel, vi tydelig beskriver trinnene som kreves for å tredimensjonalt kultur tilhenger 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

Tradisjonell, todimensjonale cellekultur teknikker ofte resultere i endrede egenskaper med hensyn til differensiering markører, cytokiner og vekstfaktorer. Tredimensjonal cellekultur i den roterende cellekultur system (RCCS) gjenoppretter uttrykk for mange av disse faktorene som vist her med en extravillous trophoblast cellelinje.

Roterende Cell Kultur Systems for Human Cell Culture: Human trophoblast Cells som en modell

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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