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 arbejde understøttes af NHMRC Program Grant # 568.969 (PSS) og Faculty of Medicine, University of New South Wales, Stem Cell Initiative (KSS).

Alle de nedenstående procedurer udføres under aseptiske inde i et klasse II biosikkerhed kabinet. Reagenser og udstyr, der anvendes, er anført i nedenstående tabeller. 1. Fremstillinq af 1,1% alginat (w / v) <ol><li> Tilsættes 0,275 g renset natriumalginat (højt glucuronsyre indhold ≥ 60%, viskositet&gt; 200 mPa s, og endotoxin ≤ 100 EU / g) i et sterilt 50 ml rør, og der tilsættes 25 ml sterilt 0,1% gelatine-opløsning fremstillet tidligere (0,5 g gelatin/500 ml milli-Q H...

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	<li>Chayosumrit, M., Tuch, B., Sidhu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alginate+microcapsule+for+propagation+and+directed+differentiation+of+hESCs+to+definitive+endoderm.">Alginate microcapsule for propagation and directed differentiation of hESCs to definitive endoderm.</a> Biomaterials. 31, 505-514 (2010).</li><li>Dean, S. K., Yulyana, Y., Williams, G., Sidhu, K. S., Tuch, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+encapsulated+embryonic+stem+cells+after+transplantation.">Differentiation of encapsulated embryonic stem cells after transplantation.</a> Transplantation. 82, 1175-1184 (2006).</li><li>Siti-Ismail, N., Bishop, A. E., Polak, J. M., Mantalaris, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+benefit+of+human+embryonic+stem+cell+encapsulation+for+prolonged+feeder-free+maintenance.">The benefit of human embryonic stem cell encapsulation for prolonged feeder-free maintenance.</a> Biomaterials. 29, 3946-3952 (2008).</li><li>Dawson, E., Mapili, G., Erickson, K., Taqvi, S., Roy, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterials+for+stem+cell+differentiation.">Biomaterials for stem cell differentiation.</a> Adv. Drug Deliv. Rev. 60, 215-228 (2008).</li><li>Orive, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+encapsulation:+promise+and+progress.">Cell encapsulation: promise and progress.</a> Nat. Med. 9, 104-107 (2003).</li><li>Watanabe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+ROCK+inhibitor+permits+survival+of+dissociated+human+embryonic+stem+cells.">A ROCK inhibitor permits survival of dissociated human embryonic stem cells.</a> Nat. Biotechnol. 25, 681-686 (2007).</li><li>Dang, S. M., Gerecht-Nir, S., Chen, J., Itskovitz-Eldor, J., Zandstra, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled,+scalable+embryonic+stem+cell+differentiation+culture.">Controlled, scalable embryonic stem cell differentiation culture.</a> Stem Cells. 22, 275-282 (2004).</li><li>Drukker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+expression+of+MHC+proteins+in+human+embryonic+stem+cells.">Characterization of the expression of MHC proteins in human embryonic stem cells.</a> Proc. Natl. Acad. Sci. U S A. 99, 9864-9869 (2002).</li><li>Calafiore, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+Technical+Procedures+for+Microencapsulation+of+Human+Islets+for+Graft+into+Nonimmunosuppressed+Patients+With+Type+1+Diabetes+Mellitus.">Standard Technical Procedures for Microencapsulation of Human Islets for Graft into Nonimmunosuppressed Patients With Type 1 Diabetes Mellitus.</a> Transplantation Proceedings. 38, 1156-1157 (2006).</li><li>Cho, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+and+large-scale+generation+of+functional+dopamine+neurons+from+human+embryonic+stem+cells.">Highly efficient and large-scale generation of functional dopamine neurons from human embryonic stem cells.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 3392-3397 (2008).</li><li>Vazin, T., Chen, J., Lee, C. T., Amable, R., Freed, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+stromal-derived+inducing+activity+in+the+generation+of+dopaminergic+neurons+from+human+embryonic+stem+cells.">Assessment of stromal-derived inducing activity in the generation of dopaminergic neurons from human embryonic stem cells.</a> Stem Cells. 26, 1517-1525 (2008).</li></ol>

Flere undersøgelser ved hjælp af musen embryonale stamceller og embryonale har vist fordelene ved 3D kultur system i biomaterialer og vævsmanipulering 2,3. Vi anvendte calciumalginatperler mikrokapsler som en egnet 3D platform til at studere embryonale formering og differentiering i sammenligning med barium alginat, idet embryonale signifikant højere levedygtighed, når indkapslet i calciumalginat end barium alginat. Denne kultur Systemet tillader også en høj densitet cellekultur og udveksling af nærin...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td><td> Noter </td></tr><tr><td> Alginat (Pronova UP MVG) </td><td> NovoMatrix </td><td> 4200101 </td><td> højt glucuronsyre indhold ≥ 60%, viskositet&gt; 200 mPa s, og endotoxin &lt;100 EU / g </td></tr><tr><td> Gelatine </td><td> Sigma-Aldrich </td><td> G1890-100G </td><td></td></tr><tr><td> 0,9% NaCI </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 sprøjtepumpe </td><td> New Era Pump Systems Inc </td><td> Model NE-1000 </td><td></td></tr><tr><td> Ezi-Flow Medical Flowmeter </td><td> Gascon Systems</td><td> G0149 </td><td></td></tr><tr><td> Y-27.632 </td><td> Merck </td><td> 688000 </td><td></td></tr><tr><td> Human 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 Udskiftning </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 Løsning </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 neural differentiering medium (basal) </td></tr><tr><td> Knockout Serum Udskiftning </td><td> Invitrogen </td><td> 10828-028 </td><td> For DA neural differentiering medium (10%) </td></tr><tr><td> Natriumpyruvat </td><td> Invitrogen </td><td> 11360070 </td><td> For DA neural differentiering medium (1 mM) </td></tr><tr><td> MEM NEAA Løsning </td><td> Invitrogen </td><td> 11140-050 </td><td> For DA nEURAL differentiering medium (0,1 mM) </td></tr><tr><td> β-mercaptoethanol </td><td> Invitrogen </td><td> 21985-023 </td><td> For DA neural differentiering medium (0,1 mM) </td></tr><tr><td> Sonic hedgehog (SHH) </td><td> R &amp; D Systems </td><td> 1314-SH-025/CF </td><td> For DA neural differentiering (100 ng / ml) </td></tr><tr><td> Fibroblastvækstfaktor 8a (FGF8a) </td><td> R &amp; D Systems </td><td> 4745-F8-050 </td><td> For DA neural differentiering (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 (embryonale) dukker op som et attraktivt alternativ kilde til celler substitutionsterapi, eftersom de kan ekspanderes i kultur på ubestemt tid og differentieres til alle celletyper i kroppen. Forskellige typer af biomaterialer er også blevet anvendt i stamcellekulturer at tilvejebringe et mikromiljø efterligne stamcelle niche 1-3. Sidstnævnte er vigtigt for at fremme celle-til-celle-interaktion, celleproliferation og differentiering i specifikke cellelinier såvel som væv organisering ved at tilvejebringe en tredimensional (3D) miljø 4, såsom indkapsling. Princippet om celleindkapsling involverer indfangning af levende celler inden for rammerne af semi-permeable membraner i 3D kulturer 2. Disse membraner tillader udveksling af næringsstoffer, oxygen og stimuli gennem membranerne, mens antistoffer og immunceller fra værten, der er større end kapslen porestørrelsen er udelukket 5. Her har vi på forhåndsendte en tilgang til kultur og differentiere embryonale DA neuroner i en 3D-mikromiljø hjælp alginatmikrokapsler. Vi har ændret dyrkningsbetingelser 2 for at øge rentabiliteten af indkapslet embryonale. Vi har tidligere vist, at tilsætning af p160-Rho-associeret coiled-coil-kinase (klippe) inhibitor, Y-27.632 og human føtal fibroblast-konditioneret serum udskiftning medium (HFF-CM) til 3D platformen signifikant forøget overlevelse af indkapslet embryonale hvor cellerne udtrykte endelige endoderm markørgener 1. Vi har nu brugt denne 3D-platform for udbredelsen af ​​embryonale og effektiv differentiering til DA neuroner. Protein og genekspression analyser efter den sidste fase af DA neuronal differentiering viste en forøget ekspression af tyrosinhydroxylase (TH), en markør for DA neuroner,&gt; 100 folds efter 2 uger. Vi antager, at vores 3D-platform ved hjælp af alginatmikrokapsler kan være nyttigt at undersøge spredningen og instrueret differentieringaf embryonale til forskellige afstamninger. Dette 3D Systemet tillader også separation af foderceller fra embryonale under processen differentiering og har også potentiale til immun-isolering under transplantation 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 optimeret en mikroindkapslingsteknik som en effektiv 3D-platform for udbredelse og differentiering af embryonale stamceller til endoderm og dopaminerge (DA) neuroner. Det giver også mulighed for immun-isolering af celler fra værten under transplantation. Platformen kan tilpasses til andre celletyper.

Alginat mikrokapselsammensætning som en 3D-platform for Formering og differentiering af humane embryonale stamceller (embryonale) til forskellige linier

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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Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

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We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

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In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

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Single-Cell Optisk Action Potentiel måling i human induceret pluripotente stamcelle-afledte kardiomyocytter

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En kultur metode til at opretholde hvilende humane hæmatopoietiske stamceller

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...

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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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As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. ...

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