Name: electric field-controlled directed migration of neural progenitor cells in 2d and 3d environments
Uploaded: 2012-02-16T12:00:00
Description: Watch this Scientific Journal Video about Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments at JoVE.com

Dette arbeidet ble støttet av Royal Society URF UF051616 stipend, Storbritannia og European Research Council STG stipend 243261 til BS. Arbeidet i MZ lab er også støttet av en California Institute of regenerativ medisin stipend RB1-01417.

1. Neural stamceller isolert <ol><li> Dissekere hele hjerner fra E14-16 mus og legg i kaldt DMEM/F12 basal medium. Fjern alle hjernehinnene under en anatomisk mikroskop og overfører hjerner inn i en 35 mm petriskål. </li><li> Bruk fine tang til mekanisk dissociate hjerner i vev fragmenter og overføre dem til en 15 ml tube, så sentrifuger prøvene ved 800 rpm i 3 min for å fjerne rusk. </li><li> Legg DMEM/F12 inneholder bFGF og EGF og triturate med en 1 ml pipette. </li><li> Pass cellesuspensjonen gjennom en celle...

<ol>
	<li>Huttenlocher, A., Horwitz, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing+with+electric+potential.">Wound healing with electric potential.</a> N. Engl. J. Med. 356, 303-303 (2007).</li><li>McCaig, C. D., Rajnicek, A. M., Song, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+cell+behavior+electrically:+current+views+and+future+potential.">Controlling cell behavior electrically: current views and future potential.</a> Physiol. Rev. 85, 943-943 (2005).</li><li>Borgens, R. B., Jaffe, L. F., Cohen, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+and+persistent+electrical+currents+enter+the+transected+lamprey+spinal+cord.">Large and persistent electrical currents enter the transected lamprey spinal cord.</a> PNAS. 77, 1209-1209 (1980).</li><li>Shapiro, S., Borgens, R., Pascuzzi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oscillating+field+stimulation+for+complete+spinal+cord+injury+in+humans:+a+phase+1+trial.">Oscillating field stimulation for complete spinal cord injury in humans: a phase 1 trial.</a> J. Neurosurg. Spine. 2, 3-3 (2005).</li><li>Zhao, M., Song, B., Pu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+signals+control+wound+healing+through+phosphatidylinositol-3-OH+kinase-gamma+and+PTEN.">Electrical signals control wound healing through phosphatidylinositol-3-OH kinase-gamma and PTEN.</a> Nature. 442, 457-457 (2006).</li><li>Yao, L., Shanley, L., McCaig, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+applied+electric+fields+guide+migration+of+hippocampal+neurons.">Small applied electric fields guide migration of hippocampal neurons.</a> J. Cell. Physiol. 216, 527-527 (2008).</li><li>Li, L., El-Hayek, Y. H., Liu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct-current+electrical+field+guides+neuronal+stem&#47;progenitor+cell+migration.">Direct-current electrical field guides neuronal stem&#47;progenitor cell migration.</a> Stem Cells. 26, 2193-2193 (2008).</li><li>Meng, X., Arocena, M., Penninger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI3K+mediated+electrotaxis+of+embryonic+and+adult+neural+progenitor+cells+in+the+presence+of+growth+factors.">PI3K mediated electrotaxis of embryonic and adult neural progenitor cells in the presence of growth factors.</a> Exp. Neurol. 227, 210-210 (2011).</li><li>Bonnici, B., Kapfhammer, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+stromal+cells+promote+neurite+extension+in+organotypic+spinal+cord+slice:+significance+for+cell+transplantation+therapy.">Bone marrow stromal cells promote neurite extension in organotypic spinal cord slice: significance for cell transplantation therapy.</a> Neurorehabil. Neural Repair. 22, 447-447 (2008).</li><li>Shichinohe, H., Kuroda, S., Tsuji, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+stromal+cells+promote+neurite+extension+in+organotypic+spinal+cord+slice:+significance+for+cell+transplantation+therapy.">Bone marrow stromal cells promote neurite extension in organotypic spinal cord slice: significance for cell transplantation therapy.</a> Neurorehabil. Neural Repair. 22, 447-447 (2008).</li><li>Song, B., Gu, Y., Pu, j. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+direct+current+electric+fields+to+cells+and+tissues+in+vitro+and+modulation+of+wound+electric+field+in+vivo.">Application of direct current electric fields to cells and tissues in vitro and modulation of wound electric field in vivo.</a> Nature Protocol. 2, 1479-1479 (2007).</li></ol>

Protokollene vi bruker er basert på tidligere studier 5,11. Ved hjelp av disse metodene, stabil kultur og elektrisk strømforhold kan opprettholdes mens søknad en EF via agar broer, Steinbergs løsning, og AG / AgCl elektroder, til celler eller skiver dyrket i custom-designede electrotactic kamre av standardiserte og presise dimensjoner. Dybden kammer kan tilpasses for ulike utvalg tykkelser 11, og i tilfelle av celler, kan kammeret størrelse bli endret for å imøtekomme imidlertid mange celler...

<table border="1"><tbody><tr><td> Navn på reagensen </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> FGF-grunnleggende Rekombinant humant </td><td> Invitrogen </td><td> PHG0026 </td><td> 20 ng / ml </td></tr><tr><td> EGF Rekombinant humant </td><td> Invitrogen </td><td> PHG0311 </td><td> 20 ng / ml </td></tr><tr><td> N2-Supplement (100X) flytende </td><td> Invitrogen </td><td> 02048 </td><td></td></tr><tr><td> DMEM/F12 medium (høy glukose) </td><td> Invitrogen </td><td> 31330-095 </td><td></td></tr><tr><td> Poly-D-Lysine </td><td> Millipore </td><td> A-003-E </td><td></td></tr><tr><td> Naturlig mus Laminin </td><td> Invitrogen </td><td> 23017-015 </td><td></td></tr><tr><td> Vekstfaktor redusert basalmembran Matrix (Matrigel) </td><td> BD Biosciences </td&gt; <td> 354230 </td><td></td></tr><tr><td> HEPES buffer </td><td> Gibco </td><td> 15630 </td><td></td></tr><tr><td> McIlwain vev chopper </td><td> Den Mickle Laboratory Engineering Co Ltd </td><td> TC752-PD </td><td></td></tr><tr><td> Dow Corning høy-vakuum silikonfett </td><td> Sigma-Aldrich </td><td> Z273554 </td><td></td></tr></tbody></table>

electric field-controlled directed migration of neural progenitor cells in 2d and 3d environments

Endogene elektriske felt (EFS) forekommer naturlig in vivo og spiller en avgjørende rolle under vev / organ utvikling og regenerasjon, herunder at den sentrale nervesystemet 1,2. Disse endogene EFS er generert av mobilnettet regulering av ionisk transport kombinert med den elektriske motstanden i celler og vev. Det har blitt rapportert som gjaldt EF behandling kan fremme funksjonell reparasjon av ryggmargsskader hos dyr og mennesker 3,4. Spesielt har EF-directed celle migrasjon blitt vist i en rekke celletyper 5,6, inkludert nevrale stamceller (NPC) 7,8. Anvendelse av likestrøm (DC) EFS er ikke en vanlig teknikk i de fleste laboratorier. Vi har beskrevet detaljerte protokoller for anvendelse av DC EFS til celle og vevskulturer tidligere 5,11. Her presenterer vi en video demonstrasjon av standard metoder basert på et beregnet feltstyrke å sette opp 2D end 3D miljøer for NPC, og å undersøke cellulære responser til EF stimulering i begge encellede vekstvilkår i 2D, og ​​organotypic ryggmargen skive i 3D. Den spinal cordslice er en ideell mottaker vev for å studere NPC ex vivo atferd, post-transplantasjon, fordi cytoarchitectonic vevet organisasjonen er godt bevart innenfor disse kulturene 9,10. I tillegg gir denne ex vivo modellen også prosedyrer som ikke er teknisk mulig å spore celler in vivo ved hjelp av time-lapse opptak på én celle nivå. Det er kritisk viktig å vurdere celle atferd i ikke bare en 2D miljø, men også i en 3D organotypic tilstand som mimicks in vivo miljøet. Dette systemet vil gi høy oppløsning med Cover glass-baserte retter i vev eller organ kultur med 3D sporing av én celle migrasjon in vitro og ex vivo og kan være en mellomliggende trinn før han går videre i VIVO paradigmer.

Watch this Scientific Journal Video about Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments at JoVE.com

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Denne protokollen demonstrerer metoder som brukes til å etablere 2D og 3D miljøer i spesialdesignede electrotactic kamre, som kan spore celler<em&gt; In vivo / ex vivo</em&gt; Med time-lapse opptak på celle-nivå, for å undersøke galvanotaxis / electrotaxis og andre cellulære responser til likestrøm (DC) elektriske felt (EFS).

Elektrisk felt-kontrollert Regissert Migrasjon av Neural stamceller i 2D og 3D miljøer

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

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