Name: alginate microcapsule as a 3d platform for propagation and differentiation of human embryonic stem cells (hesc) to different lineages
Uploaded: 2012-03-09T12:00:00
Description: Watch this Scientific Journal Video about Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages at JoVE.com

Dette arbeidet støttes av NHMRC Program Grant # 568969 (PSS) og Det medisinske fakultet, Universitetet i New South Wales, Stem Cell Initiative (KSS).

Alle prosedyrene nedenfor er utført ved hjelp av aseptisk teknikk inne i en klasse II biosikkerhet Cabinet. Reagenser og utstyr brukt, er oppført i tabellene nedenfor. 1. Utarbeidelse av 1,1% Alginat (w / v) <ol><li> Legg 0.275 g renset natrium alginate (høy glukuronsyre innhold ≥ 60%, viskositet&gt; 200 mPa s, og endotoksin ≤ 100 EU / g) i en steril 50 ml tube og tilsett 25 ml steril 0,1% gelatin løsning forberedt tidligere (0,5 g gelatin/500 ml Milli-Q H 2 O og oppløs...

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Flere studier ved hjelp av musen embryonale stamceller og hESC har demonstrert nytten av 3D kultur system i biomaterialer og tissue engineering 2,3. Vi brukte kalsiumalginatpinner mikrokapsler som et egnet 3D plattform for å studere hESC forplantning og differensiering i forhold til barium alginat siden hESC viste signifikant høyere levedyktighet når innkapslet i kalsiumalginatpinner enn barium alginat. Denne kulturen Systemet tillater også en høy tetthet cellekultur og utveksling av næringsstoffer og o...

<table border="1"><tbody><tr><td> Navn på reagensen </td><td> Firma </td><td> Katalognummer </td><td> Merknader </td></tr><tr><td> Alginat (Pronova OPP MVG) </td><td> NovoMatrix </td><td> 4200101 </td><td> høyt glukuronsyre innhold ≥ 60%, viskositet&gt; 200 mPa s, og endotoksin &lt;100 EU / g </td></tr><tr><td> Gelatin </td><td> Sigma-Aldrich </td><td> G1890-100G </td><td></td></tr><tr><td> 0,9% NaCl </td><td> Baxter Healthcare </td><td> AHF7123 </td><td></td></tr><tr><td> Type J1 perle generator </td><td> Nisco Engineering Inc </td><td> SPA-0447 </td><td></td></tr><tr><td> Multi-Phaser Sprøytepumpe </td><td> New Era Pump Systems Inc </td><td> Modell NE-1000 </td><td></td></tr><tr><td> Ezi-Flow Medical strømningsmåler </td><td> Gascon Systems</td><td> G0149 </td><td></td></tr><tr><td> Y-27632 </td><td> Merck </td><td> 688000 </td><td></td></tr><tr><td> Humant serumalbumin </td><td> Sigma-Aldrich </td><td> A4327-1G </td><td></td></tr><tr><td> Accutase </td><td> Millipore </td><td> SCR005 </td><td></td></tr><tr><td> 14g x 2 &quot;IV kateter </td><td> TERUMO </td><td> SR-OX1451C </td><td></td></tr><tr><td> Knockout-DMEM </td><td> Invitrogen </td><td> 10829-018 </td><td> For SR medium (basal) </td></tr><tr><td> GlutaMAX-I </td><td> Invitrogen </td><td> 35050-061 </td><td> For SR medium (2 mm) </td></tr><tr><td> Knockout Serum Replacement </td><td> Invitrogen </td><td> 10828-028 </td><td> For SR medium (20%) </td></tr><tr><td> Penicillin-Streptomycin </td><td> Invitrogen </td><td> 15070063 </td><td> For SR medium (2,5 U / ml) </td&gt; </tr><tr><td> Insulin-Transferrin-Selen (ITS) </td><td> Invitrogen </td><td> 41400045 </td><td> For SR medium (1x) </td></tr><tr><td> β-Mercaptoethanol </td><td> Invitrogen </td><td> 21985-023 </td><td> For SR medium (0,1 mm) </td></tr><tr><td> MEM NEAA Solution </td><td> Invitrogen </td><td> 11140-050 </td><td> For SR medium (5 mm) </td></tr><tr><td> Glasgow Minimum Essential Medium </td><td> Invitrogen </td><td> 11710035 </td><td> For DA nevrale differensiering medium (basal) </td></tr><tr><td> Knockout Serum Replacement </td><td> Invitrogen </td><td> 10828-028 </td><td> For DA nevrale differensiering medium (10%) </td></tr><tr><td> Sodium pyruvat </td><td> Invitrogen </td><td> 11360070 </td><td> For DA nevrale differensiering medium (1 mm) </td></tr><tr><td> MEM NEAA Solution </td><td> Invitrogen </td><td> 11140-050 </td><td> For DA nEURAL differensiering medium (0,1 mm) </td></tr><tr><td> β-Mercaptoethanol </td><td> Invitrogen </td><td> 21985-023 </td><td> For DA nevrale differensiering medium (0,1 mm) </td></tr><tr><td> Sonic hedgehog (ssh) </td><td> R &amp; D Systems </td><td> 1314-SH-025/CF </td><td> For DA nevrale differensiering (100 ng / ml) </td></tr><tr><td> Fibroblast vekstfaktor 8a (FGF8a) </td><td> R &amp; D Systems </td><td> 4745-F8-050 </td><td> For DA nevrale differensiering (100 ng / ml) </td></tr></tbody></table>

alginate microcapsule as a 3d platform for propagation and differentiation of human embryonic stem cells (hesc) to different lineages

Humane embryonale stamceller (hESC) er fremstår som et attraktivt alternativ kilde for celle erstatning terapi siden de kan bli utvidet i kultur ubestemt tid og differensiert til eventuelle celletyper i kroppen. Ulike typer biomaterialer har også blitt brukt i stamcelle kulturer for å gi en mikromiljøet etterligne stamcelleforskningen nisje 1-3. Sistnevnte er viktig for å fremme celle-til-celle interaksjon, celleproliferasjon og differensiering inn i spesifikke linjene samt vev organisering ved å tilby en tredimensjonal (3D) miljø 4 som innkapsling. Prinsippet om cellen innkapsling innebærer entrapment av levende celler innenfor rammen av semi-permeable membraner i 3D kulturer 2. Disse membranene tillater utveksling av næringsstoffer, oksygen og stimuli på tvers av membraner, mens antistoffer og immunceller fra verten som er større enn kapselen porestørrelse er ekskludert fem. Her forhåndsinstallert visendte en tilnærming til kultur og differensiere hESC DA nevroner i en 3D mikromiljøet hjelp alginat mikrokapsler. Vi har endret de kultur vilkår 2 for å forbedre levedyktigheten til innkapslede hESC. Vi har tidligere vist at tillegg av P160-Rho-assosiert coiled-coil kinase (ROCK) hemmer, Y-27632 og human føtal fibroblast-condition serum erstatning medium (HFF-CM) til 3D-plattformen betydelig forbedret levedyktighet innkapslede hESC der cellene uttrykte definitive endoderm markør gener en. Vi har nå brukt denne 3D plattform for spredning av hESC og effektiv differensiering til DA nerveceller. Protein og genekspresjon analyser etter den siste etappen av DA neuronal differensiering viste en økt uttrykk av tyrosin hydroksylase (TH), en markør for DA nevroner,&gt; 100 folder etter 2 uker. Vi hypotesen at vår 3D plattform ved hjelp av alginat mikrokapsler kan være nyttig å studere spredning og regissert differensieringav hESC til ulike linjene. Denne 3D Systemet tillater også separasjonen av materen celler fra hESC under prosessen for differensiering, og har også potensial for immun-isolering under transplantasjon i fremtiden.

Watch this Scientific Journal Video about Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages at JoVE.com

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

Vi har optimalisert en microencapsulation teknikk som en effektiv 3D plattform for spredning og differensiering av embryonale stamceller til endoderm og dopaminerge (DA) nerveceller. Det gir også en mulighet for immun-isolering av celler fra verten i transplantasjon. Denne plattformen kan tilpasses andre celletyper.

Alginat Microcapsule som en 3D-plattform for Formering og Differensiering av humane embryonale stamceller (hESC) til ulike linjene

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

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Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

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

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The blood-brain barrier (BBB) is a key challenge for the successful delivery of drugs to the brain. Ultrasound exposure in the presence of microbubbles ...

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