Name: rapid fibroblast removal from high density human embryonic stem cell cultures
Uploaded: 2012-10-28T16:00:00
Description: Watch this Scientific Journal Video about Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures at JoVE.com

Dette arbeidet ble finansiert av en New fakultet Award II fra California Institute of regenerativ medisin (RN2-00921-1), og en NIH-finansiert National Research Award (F32-HL104924)

1. Utarbeidelse av mus embryonale Feeders <ol><li> Den MEFs brukt i co-kultur av hESCs bør tidligere behandlet ved bestråling eller Mitomycin-C for å inhibere cellene fra gjennomgår divisjon. </li><li> To timer før MEF seeding, pels hver 60 mm plasma behandlet kultur tallerken med 2 ml 0,05% Gelatin. </li><li> Tine en ampulle med behandlet MEFs og plate på ~ 20000 celler / cm 2. </li><li> La cellene å overholde natten. </li></ol> 2. Co-seeding hESCS bort på MEFs <ol><li> Ny...

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Metoden presentert i dette manuskriptet tilbyr rask og mindre kostnadskrevende alternativ til eliminere fibroblast mater celler fra humane embryonale cellekulturer. Vellykket fjerning av fibroblaster er avhengig av eksistensen av en tett konfluent monolag av disse cellene som utvikler seg i lengre stilk cellekulturer. Etter 7-10 dager vil den voksende hESC kolonier skyve mater fibroblaster i en ytre retning, genererer en stadig tett fibroblast monolayer mellom koloniene. De sterke celle-til-celle vedlegg i fibroblast mo...

<table border="1"><tbody><tr><td> Navnet på reagensen </td><td> Selskapet </td><td> Katalognummer </td><td> Kommentarer (valgfritt) </td></tr><tr><td> DDR2 </td><td> Santa Cruz </td><td> 7555 </td><td> Fibroblast Marker </td></tr><tr><td> Dapi </td><td> Calbiochem </td><td> 268298 </td><td> Cellekjernen Marker </td></tr><tr><td> SSEA-4 </td><td> Millipore </td><td> MAB4304 </td><td> Human ESC Marker </td></tr><tr><td> Oct 3/4 </td><td> Santa Cruz </td><td> 9081 </td><td> Human ESC Marker </td></tr><tr><td> FLK-1 </td><td> BD Pharma </td><td> 555307 </td><td> Tidlig Differensiering Marker </td></tr><tr><td> Tra-1-81 </td><td> Acris </td><td> AM20377AF4-S </td><td> Human ESC Marker </td></tr></tbody></table> Tabell 1. Tabell over spesiell grunngents brukes til farging av hESC.

rapid fibroblast removal from high density human embryonic stem cell cultures

Mus embryonale fibroblaster (MEFs) ble brukt til å etablere menneskelige embryonale stamceller (hESCs) kulturer etter blastocyst isolasjon en. Dette matesystem opprettholder hESCs fra gjennomgår spontan differensiering under celle ekspansjon. Imidlertid er denne co-dyrkning arbeidsintensiv, krever høyt utdannet personell, og gir nede hESC renhet 4. Mange laboratorier har forsøkt å minimere antall mater celler i hESC kulturer (dvs. innlemme matrise-belagte retter eller andre mater celletyper 5-8). Disse modifiserte kulturer har vist noen lover, men har ikke fortrengt standard metode for dyrking hESCs med mitomycin C-behandlede mus embyronic fibroblaster for å forsinke uønsket spontan differensiering av hESC kulturer. Derfor bør materen celler brukes i hESC ekspansjon fjernes under differensiering eksperimenter. Selv om flere teknikker er tilgjengelige for å rense hESC colonies (FACS, Mac, eller bruk av resistente vektorer) fra feeders, disse teknikkene er arbeidskrevende, kostbar og / eller ødeleggende for hESC. Målet med dette prosjektet var å oppfinne en metode for rensing som gjør at høsting av en renere befolkning på hESCs. Vi har observert at i en konfluent hESC kultur, kan MEF befolkningen fjernes med en enkel og rask aspirasjon av MEF arket. Denne fjerningen er avhengig av flere faktorer, inkludert lateral celle-til-celle binding av MEFs som har en lavere bindingsaffinitet til styren kultur parabol, og evnen av stamcelle koloniene å presse fibroblaster utover under generering av sin egen &quot; nisje &quot;. Den hESC ble deretter undersøkt for SSEA-4, Oct3 / 4 og Tra 1-81 uttrykk opptil 10 dager etter MEF fjerning å sikre opprettholdelsen av pluripotency. Videre var hESC kolonier i stand til å fortsette å vokse fra i større formasjoner etter MEF fjerning, som gir et ekstra nivå av hESC ekspansjon.

Watch this Scientific Journal Video about Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures at JoVE.com

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures (Video) | JoVE

Til tross for pågående arbeidet med å overgangen kulturer til mater-frie forhold, avledning og kultur av menneskelige embryonale stamceller (hESC) fortsatt i stor grad avhengig av co-kulturer med mus embryonale feeders (MEFs). Her viser vi en ny metodikk for raskt å fjerne feeders fra hESC kulturer før eksperimentering.

Rapid Fibroblast fjerning fra High Density Menneskelig embryonale stamceller kulturer

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system ...

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Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

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