Name: primary culture of human vestibular schwannomas
Uploaded: 2014-07-20T14:00:00
Description: Watch this Scientific Journal Video about Primary Culture of Human Vestibular Schwannomas at JoVE.com

Support: NIDCD R01DC009801, P30DC010362, 5T32DC000040-17

Etikk Uttalelse: bruk av de menneskelige vevsprøver i denne protokollen ble godkjent av University of Iowa Institutional Review Board (IRB). En. Oppsett dagen før Tumor Harvests <ol><li> Coat Cell Kultur Plater: I en steril kultur hette, legger nok poly-L-ornithine (0,01% løsning) til å dekke bunnen av en isopor / plast kultur godt / parabol, erstatte lokkene, og la sitte i romtemperatur i minst 2 timer. Glass lysbilder eller dekkglass kan erstatte isopor for eksperimenter som krever b...

<ol>
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Historie av in vitro schwannoma kulturer Arbeidet med å etablere in vitro schwannoma kulturer begynte bare et par tiår etter den første Vestibularisschwannom åpen kirurgisk reseksjon skjedde i 1894 12. Den første registrerte kulturer var ved Kredel på 1920-tallet, som uten hell forsøkt å vokse hakket svulsten ved &quot;hengende slipp-metoden&quot; 12 . Senere, Drs. Murray og Stout publisert sin in vitro kultur erfaring...

Vestibular schwannomas (VSS) representerer Schwann celle (SC) svulster i vestibular nerve, at det går 10% av alle intrakranielle svulster 1-3. VSS oppstå i enten sporadisk eller familiære (Nevrofibromatose type 2, NF2) former, både knyttet til inaktive defekter i NF2 tumor suppressor gen. Behandling for VSS er generelt kirurgisk reseksjon eller radiosurgery, men sykelighet av slike prosedyrer inkluderer døvhet, ansikts nevropatier, spinalvæske lekkasje, ubalanse, og svulst gjenvekst en. I tillegg er ikke alle pasienter er akseptable kirurgisk eller stråle kandidater. En slik betydelig sykelighet samt en mangel på alternative behandlingsformer har drevet etterforskning i den unike molekylærbiologi av VSS i håp om å utvikle nye behandlinger 3,4. Cellekultur gir mulighet for raske, lettvinte, og dyptgående analyse av molekylære og cellulære atferd og screening av potensielle terapeutiske compounds. Historisk sett har manglende tilgang til ferske vevsprøver og det faktum at schwannoma cellene ikke blir foreviget vesentlig hemmet bruk av primærkulturer for etterforskning av schwannoma tumorigenesis. For å overvinne den begrensede tilførsel av primærkulturer, ble de udødelig HEI193 VS cellelinje generert ved transduksjon med HPV E6 og E7 onkogener 5. Det onkogene transduksjon innført betydelige molekylære og fenotypiske endringer i cellene, noe som begrenser deres anvendelse som en modell for human schwannoma tumorer. SC kulturer som stammer fra transgene mus linjer som mangler et funksjonelt NF2-genet representerer et annet alternativ for å undersøke NF2-avhengige SC tumorigenesis in vitro. Disse kulturene derimot ikke klarer å rekapitulere den heterogene natur menneskelig VSS eller konto for menneskelig bestemt atferd. De fleste tidligere VS kultur teknikker som kreves relativt lang saksbehandlingstid og kompliserte selektive kultur teknikker 6-8.Her presenterer vi en enkel, reproduserbar protokoll for primær VS cellekultur med full prosessering på under tre timer, med 95% tumorcelle renhet som bestemmes av farging.

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			<td height="42" style="height:42px;width:216px;">Poly-L-Ornithine, 0.01% Solution</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">P4957</td>
			<td style="width:167px;">Cell Culture Surface Treatment</td>
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			<td height="42" style="height:42px;width:216px;">Laminin Mouse Protein&#160;</td>
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			<td style="width:71px;">Sigma</td>
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			<td>Dissociation Reagent</td>
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			<td>Dissociation Reagent</td>
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			<td height="21" style="height:21px;width:216px;">Small Forceps</td>
			<td style="width:71px;">FST</td>
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			<td style="width:71px;">FST</td>
			<td style="width:119px;">10004-13</td>
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			<td height="21" style="height:21px;width:216px;">#11 Scalpel Blade</td>
			<td style="width:71px;">Roboz</td>
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			<td height="42" style="height:42px;width:216px;">Non-Tissue Culture 100mm Round Petri Dish</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">50-820-904</td>
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			<td height="42" style="height:42px;width:216px;">P1000 Pipetteman</td>
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			<td height="42" style="height:42px;width:216px;">Serological Pipetteman</td>
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			<td>Schwannoma Culture and Media Components</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hanks Balanced Salt Solution (HBSS) -/- (w/out Ca2+, Mg2+)</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">14170-112</td>
			<td>Schwannoma Culture and Media Components</td>
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			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">11965-092</td>
			<td>Schwannoma Culture and Media Components</td>
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			<td style="width:71px;">Gibco</td>
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			<td>Schwannoma Culture and Media Components</td>
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			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">26140-079</td>
			<td>Schwannoma Culture and Media Components</td>
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			<td style="width:71px;">Gibco</td>
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			<td>Schwannoma Culture and Media Components</td>
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			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">I6634</td>
			<td>Schwannoma Culture and Media Components</td>
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primary culture of human vestibular schwannomas

Vestibular schwannomas (VSS) representerer Schwann celle (SC) svulster i vestibular nerve, at det går 10% av alle intrakranielle svulster. VSS oppstå i enten sporadisk eller familiære (Nevrofibromatose type 2, NF2) former, både knyttet til inaktive defekter i NF2 tumor suppressor gen. Behandling for VSS er generelt kirurgisk reseksjon eller radiosurgery, men sykelighet av slike prosedyrer har drevet undersøkelser i mindre invasive behandlinger. Historisk sett har manglende tilgang til ferske vevsprøver og det faktum at schwannoma cellene ikke blir foreviget vesentlig hemmet bruk av primærkulturer for etterforskning av schwannoma tumorigenesis. For å overvinne den begrensede tilførsel av primærkulturer, ble de udødelig HEI193 VS cellelinje generert ved transduksjon med HPV E6 og E7 onkogener. Det onkogene transduksjon innført betydelige molekylære og fenotypiske endringer i cellene, noe som begrenser deres anvendelse som en modell for human schwannoma svulster. Vi derfor illustrere en forenklet, reproduserbar protokoll for kultur av primær menneskelig VS celler. Dette lett mestret teknikken gjør det mulig for molekylære og cellulære undersøkelser som mer nøyaktig rekapitulere kompleksiteten i VS sykdom.

Riktig tumor fragmentering er viktig for optimal såing og utvekst i vevkulturskåler (figur 1). Identifisering av schwannoma celler i løpet av de første dagene etter plating er ofte vanskelig på grunn av obscuring mengder av celle-debris, og røde blodceller. Ved 4. eller 5. dag, vil celle utvidelser nærheten heft tumor fragmenter skape gjenkjennelige &#39;snøring&#39; mønstre blant vrakrestene. Påfølgende medier endringer generelt fjerne mesteparten av ikke-adherente cell...

Watch this Scientific Journal Video about Primary Culture of Human Vestibular Schwannomas at JoVE.com

Primary Culture of Human Vestibular Schwannomas

Primær Culture of Human Vestibulær schwannomas

Vestibulær schwannomas (VSS) er ikke-maligne svulster av Schwann celle (SC) opprinnelse, assosiert med mutasjoner i NF2 tumorsuppressorgen. Vi rapporterer en reproduserbar, effektiv protokoll til primære humane VS cellekultur som gjør det mulig for molekylær-og celle eksperimentell manipulering og analyse og sammenfatter den heterogene natur sykdom hos mennesker.

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in ...

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