Name: generation of organotypic raft cultures from primary human keratinocytes
Uploaded: 2012-02-22T13:00:00
Description: Watch this Scientific Journal Video about Generation of Organotypic Raft Cultures from Primary Human Keratinocytes at JoVE.com

Forfatterne vil gerne takke Sally Roberts (University of Birmingham, Birmingham UK) for den slags gave E1E4 antistof. Dette arbejde blev støttet af en bevilling fra National Cancer Institute (4R00CA137160-03).

1. Forberedelse til Organotypiske Raft Cultures <ol><li> Metal flåder gitre meget først behandles med chromsyre svovlsyre for at fjerne enhver rest, som kan interferere med differentieringsprocessen. Nedsænkes metalgitre i et bægerglas indeholdende svovlsyre i en time, derefter kontinuerligt skyllet natten over med ledningsvand. Efter natten skylles, bør tømmerflåde net skal skylles i 3-5 timer i dobbelt destilleret vand. </li><li> At yde støtte til de flåder, bøjer tre sider af flåden omkring 0,5 cm i samm...

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	<li>Andrei, G., Duraffour, S., VandenOord, J., Snoeck, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+raft+cultures+for+investigations+of+virus+growth,+pathogenesis+and+efficacy+of+antiviral+agents.">Epithelial raft cultures for investigations of virus growth, pathogenesis and efficacy of antiviral agents.</a> Antiviral Research. 85, 431-449 (2010).</li><li>Asselineau, D., Prunieras, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+'simplified'+skin:+control+of+fabrication.">Reconstruction of 'simplified' skin: control of fabrication.</a> The British Journal of Dermatology. 111, 219-222 (1984).</li><li>Bodily, J., Alam, S., Chen, H., Meyers, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Organotypic epithelial raft cultures and the study of the natural history of human papillomavirus. , (2006).</li><li>Cheng, S., Schmidt-Grimminger, D. C., Murant, T., Broker, T. R., Chow, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation-dependent+up-regulation+of+the+human+papillomavirus+E7+gene+reactivates+cellular+DNA+replication+in+suprabasal+differentiated+keratinocytes.">Differentiation-dependent up-regulation of the human papillomavirus E7 gene reactivates cellular DNA replication in suprabasal differentiated keratinocytes.</a> Genes &#38; Development. 9, 2335-2349 (1995).</li><li>Dollard, S. C., Wilson, J. L., Demeter, L. M., Bonnez, W. R., Reichman, C., Broker, T. R., Chow, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+human+papillomavirus+and+modulation+of+the+infectious+program+in+epithelial+raft+cultures.+OFF.">Production of human papillomavirus and modulation of the infectious program in epithelial raft cultures. OFF.</a> Genes &#38; Development. 6, 1131-1142 (1992).</li><li>Frattini, M. G., Lim, H. B., Laimins, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+synthesis+of+oncogenic+human+papillomaviruses+requires+episomal+genomes+for+differentiation-dependent+late+expression.">In vitro synthesis of oncogenic human papillomaviruses requires episomal genomes for differentiation-dependent late expression.</a> Proceedings of the National Academy of Sciences of the United States of America. 93, 3062-3067 (1996).</li><li>Kopan, R., Traska, G., Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoids+as+important+regulators+of+terminal+differentiation:+examining+keratin+expression+in+individual+epidermal+cells+at+various+stages+of+keratinization.">Retinoids as important regulators of terminal differentiation: examining keratin expression in individual epidermal cells at various stages of keratinization.</a> The Journal of Cell Biology. 105, 427-440 (1997).</li><li>Longworth, M. S., Laimins, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+human+papillomaviruses+in+differentiating+epithelia.">Pathogenesis of human papillomaviruses in differentiating epithelia.</a> Microbiology and Molecular Biology Review. 68, 362-372 (2004).</li><li>Meyers, C., Frattini, M. G., Hudson, J. B., Laimins, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosynthesis+of+human+papillomavirus+from+a+continuous+cell+line+upon+epithelial+differentiation.">Biosynthesis of human papillomavirus from a continuous cell line upon epithelial differentiation.</a> Science. 257, 971-973 (1992).</li><li>Wilson, R., Laimins, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Differentiation of HPV-Containing Cells Using Organotypic Raft Culture or Methylcellulose. , 119-119 (2006).</li><li>zur Hausen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Papillomavirus+infections--a+major+cause+of+human+cancers.">Papillomavirus infections--a major cause of human cancers.</a> Biochimica et Biophysica Acta. 1288, F55-F78 (1996).</li></ol>

Vi beskriver her en fremgangsmåde, som kan anvendes til at studere epitheldifferentiering i almindelighed, men kan også let tilpasses til at studere viral patogenese, såvel som effekten af ​​potentielle terapeutiske midler. Mange virus målrette epitelceller enten det primære sted for infektion, som i tilfælde af HPV eller på et tidspunkt i den virale livscyklus, som med herpesvirus. Skønt stigende organotypiske flåder kulturer er tidskrævende, evnen til nøjagtigt rekapitulere in vivo epitheldiffe...

<table border="1"><tbody><tr><td> Navn reagens </td><td> Firma </td><td> Katalog nr. </td></tr><tr><td> Rotte Tail Collagen type 1 (4-5mg/ml) </td><td> BD Biosciences </td><td> 354236 </td></tr><tr><td> DMEM uden NaHC0 3 </td><td> Invitrogen </td><td> 12100-061 </td></tr><tr><td> NaHC0 3 </td><td> Sigma </td><td> S5761 </td></tr><tr><td> Hepes </td><td> Calbiochem </td><td> 391338 </td></tr><tr><td> Rustfrit stål metalgitre </td><td> Williams og fyrighed Co </td><td> CR-03063-040-100-S </td></tr><tr><td> E-medium 11 </td><td></td><td></td></tr><tr><td> Epidermal Growth Factor </td><td> BD Biosciences </td><td> 354010 </td></tr></tbody></table>

generation of organotypic raft cultures from primary human keratinocytes

Udviklingen af organotypiske epitel flåder kulturer har givet forskere med en effektiv in vitro system, der nøjagtigt gengiver epitheldifferentiering. Der er mange anvendelser for dette system. For eksempel, har evnen til at vokse tre-dimensionelle organotypiske tømmerflåde kulturer af keratinocytter været en vigtig milepæl i studiet af human papillomavirus (HPV) 1. Livscyklus HPV er tæt forbundet med differentiering af skæl-epitel 2. Organotypiske epitel tømmerflåde kulturer, som det fremgår her gengiver hele papillomavirus livscyklus, herunder virusproduktion 3,4,5. Desuden udviser disse flåder kulturer dysplastiske læsioner svarende til dem observeret ved in vivo-infektion med HPV. Derfor dette system kan også anvendes til at studere epitelcelletyper cancere, såvel som virkningen af ​​lægemidler på epitel celledifferentiering i almindelighed. Oprindeligt udviklet af Asselineau og Prunieras <sop&gt; 6 og modificeret af Kopan et al. 7 er organotypisk epitel redningsflåden dyrkningssystem modnet i et generelt relativt let kultur model, som involverer dyrkning af celler på collagen sættes holdt ved en luft-væske-grænsefladen (figur 1A). Løbet af 10-14 dage stratificere cellerne og differentierer til dannelse af en fuld tykkelse epithel, der producerer differentiering-specifikke cytokeratiner. Høstede klumper kan undersøges histologisk, såvel som ved standard molekylære og biokemiske teknikker. I denne artikel beskriver vi en fremgangsmåde til generering af flåder kulturer fra primære humane keratinocytter. Den samme teknik kan anvendes med etablerede epiteliale cellelinjer, og kan nemt tilpasses til brug med epitelvæv fra normale eller syge biopsier 8. Mange vira målgrupper enten kutan eller slimhindeepithelet som en del af deres replikation livscyklus. I løbet af de sidste mange år er anvendelsen af ​​muligheden for organotypisk række kultninger, som en metode til at studere virus-værts-celle-interaktioner er blevet vist i adskillige herpesvira, samt adenovirus, parvovira, og poxviruser 9. Organotypiske flåder kulturer kan således tilpasses til at behandle viral patogenese, og er de eneste midler til at teste hidtil ukendte antivirale midler for de vira, der ikke kan dyrkes i permanente cellelinier.

Watch this Scientific Journal Video about Generation of Organotypic Raft Cultures from Primary Human Keratinocytes at JoVE.com

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

En<em&gt; In vitro</em&gt; Metode til at efterligne<em&gt; In vivo</em&gt; Epitheldifferentiering er beskrevet. Mange virus målrette epitelceller som en del af deres virale livscyklus, og denne fremgangsmåde tilvejebringer et middel til at undersøge virus: vært interaktioner, der mere ligner den, der forekommer<em&gt; In vivo</em&gt;. Denne teknik kan anvendes med primære keratinocytter, etablerede cellelinier, såvel som normale eller syge biopsivæv.

Generering af Organotypiske Raft Cultures fra primære humane keratinocytter

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

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