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

Etik Statement: udnyttelse af de menneskelige tumor prøver i denne protokol blev godkendt af University of Iowa Institutional Review Board (IRB). 1.. Opsætning dagen før Tumor Harvest <ol><li> Coat cellekulturplader: I en steril kultur hætte, tilføje nok poly-L-ornithin (0,01% opløsning) til at dække bunden af ​​en polystyren / plast kultur godt / skål, erstatte låg og lad stå ved stuetemperatur i mindst 2 timer. Glas dias eller dækglas kan erstatte polystyren til eksperimen...

<ol>
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Historie af in vitro Schwannoma Cultures Bestræbelser på at etablere in vitro schwannoma kulturer begyndte blot et par årtier efter den indledende akustiske neuroma åben kirurgisk resektion forekom i 1894 12. Den første registrerede kulturer var ved Kredel i 1920&#39;erne, der forgæves har forsøgt at vokse hakket tumor med &quot;hængende dråbe-metoden&quot; 12 . Senere, Drs. Murray og Stout offentliggjort deres in vitro-dy...

Vestibulære schwannomas (VSS) udgør Schwann celle (SC) tumorer i det vestibulære nerve, det går 10% af alle intrakranielle neoplasmer 1-3. VSS forekomme i enten sporadiske eller familiær (neurofibromatosis type 2, NF2) former, både i forbindelse med inaktivering defekter i NF2 tumorsuppressor gen. Behandling for VSS er generelt kirurgisk resektion eller strålebehandling, men sygelighed af sådanne procedurer inkluderer døvhed, ansigts neuropatier, lækage spinal, ubalance og tumorgenvækst 1. Derudover ikke alle patienter er acceptable kirurgisk eller stråling kandidater. En sådan betydelig sygelighed samt en mangel på alternative behandlingsformer har drevet undersøgelse af unikke molekylærbiologi VSS i håb om at udvikle nye behandlinger 3,4. Celledyrkning giver mulighed for hurtig, letkøbt og dybtgående analyse af molekylære og cellulære adfærd og screening af potentielle terapeutiske compounds. Historisk set har manglende adgang til friske vævsprøver og det faktum, at schwannoma cellerne ikke udødeliggjort hæmmet brugen af ​​primære kulturer til undersøgelse af schwannoma tumorigenese. For at overvinde det begrænsede udbud af primære kulturer, blev udødeliggjort HEI193 VS cellelinje genereret af transduktion med HPV E6 og E7 onkogener 5. Denne onkogen transduktion indført væsentlige molekylære og fænotypiske ændringer i de celler, der begrænser deres anvendelse som model for humane schwannoma tumorer. SC kulturer afledt fra transgene mus linjer, der mangler et funktionelt NF2 gen udgør et alternativ til at undersøge NF2-afhængig SC tumorigenese in vitro. Disse kulturer dog undlader at rekapitulere den heterogene karakter af menneskelig VSS eller konto for menneskelig bestemt adfærd. De fleste tidligere VS dyrkningsteknikker kræves relativt lange sagsbehandlingstider og komplicerede selektive dyrkningsteknikker 6-8.Her præsenterer vi en simpel, reproducerbar protokol til primær VS cellekultur med komplet behandling i under 3 timer, med 95% tumor celle renhed som bestemt ved immunfarvning.

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primary culture of human vestibular schwannomas

Vestibulære schwannomas (VSS) udgør Schwann celle (SC) tumorer i det vestibulære nerve, det går 10% af alle intrakranielle neoplasmer. VSS forekomme i enten sporadiske eller familiær (neurofibromatosis type 2, NF2) former, både i forbindelse med inaktivering defekter i NF2 tumorsuppressor gen. Behandling for VSS er generelt kirurgisk resektion eller strålebehandling, men sygelighed af sådanne procedurer har drevet undersøgelser mindre invasive behandlinger. Historisk set har manglende adgang til friske vævsprøver og det faktum, at schwannoma cellerne ikke udødeliggjort hæmmet brugen af ​​primære kulturer til undersøgelse af schwannoma tumorigenese. For at overvinde den begrænsede forsyning af primære kulturer blev udødeliggjort HEI193 VS cellelinje genereret ved transduktion med HPV E6-og E7 oncogener. Denne onkogen transduktion indført væsentlige molekylære og fænotypiske ændringer i de celler, der begrænser deres anvendelse som model for humane schwannoma tumorer. Vi illustrerer derfor en forenklet, reproducerbar protokol til dyrkning af primære humane VS celler. Denne nemt mestrer teknik gør det muligt for molekylære og cellulære undersøgelser, der mere præcist rekapitulere kompleksiteten af ​​VS sygdom.

Korrekt tumor fragmentering er afgørende for optimal såning og udvækst i vævskulturskåle (figur 1). Identifikation af schwannoma celler under de første dage efter udpladning er ofte vanskeligt på grund af at tilsløre mængder celleaffald og røde blodlegemer. Ved det 4. eller 5. dag, vil celle udvidelser nær vedhængende tumorfragmenter skabe genkendelige &#39;snøring&#39; mønstre blandt murbrokkerne. Efterfølgende medieændringer generelt fjerne størstedelen af ​​...

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 tumorer af Schwann celle (SC) oprindelse, der er forbundet med mutationer i NF2 tumorsuppressorgenet. Vi rapporterer en reproducerbar, effektiv protokol til primære humane VS cellekultur, der giver mulighed for molekylære og cellulære eksperimentel manipulation og analyse og samler op heterogene karakter af sygdom 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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