Name: skin punch biopsy explant culture for derivation of primary human fibroblasts
Uploaded: 2013-07-07T13:00:00
Description: Watch this Scientific Journal Video about Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts at JoVE.com

Udviklingen af ​​denne protokol blev finansieret af California Institute for regenerativ medicin (CIRM, TR1-01246) og Parkinsons Alliance.

Huden Punch biopsi opnået ved hjælp af standard procedure 3 (f.eks opnået med 4mm rund Visipunch instrument) bør holdes i fuldstændig DMEM 20% FBS medier på is. Når prøven er kommet i laboratoriet, behandle biopsi snarest muligt. 1.. Forberedelse af Skin Punch biopsi TRIN 1,1-1,3 skal udføres INSIDE A BIOSIKKERHED CABINET <ol><li> Forberede sig på forhånd: Tilsæt 1 ml 0,1% Gelatine til hver brønd i en 6-brønds ...

<ol>
	<li>Nguyen, H. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LRRK2+mutant+iPSC-derived+DA+neurons+demonstrate+increased+susceptibility+to+oxidative+stress.">LRRK2 mutant iPSC-derived DA neurons demonstrate increased susceptibility to oxidative stress.</a> Cell Stem Cell. 8, 267-280 (2011).</li><li>Mak, S. K., Tewari, D., Tetrud, J. W., Langston, J. W., Schule, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+in+skin+fibroblasts+from+a+Parkinson's+disease+patient+with+an+alpha-synuclein+triplication.">Mitochondrial dysfunction in skin fibroblasts from a Parkinson's disease patient with an alpha-synuclein triplication.</a> Journal of Parkinson's Disease. 1, 175-183 (2011).</li><li>Punch Zuber, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=biopsy+of+the+skin.">biopsy of the skin.</a> Am. Fam. Physician. 65, 1155-1164 (2002).</li><li>Takashima, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+fibroblast+cultures.">Establishment of fibroblast cultures.</a> Curr. Protoc. Cell Biol. Chapter 2, Unit 2, 1 (2001).</li><li>Kuroda, K., Tajima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HSP47+is+a+useful+marker+for+skin+fibroblasts+in+formalin-fixed,+paraffin+embedded+tissue+specimens.">HSP47 is a useful marker for skin fibroblasts in formalin-fixed, paraffin embedded tissue specimens.</a> J. Cutan. Pathol. , 241-246 (2004).</li></ol>

Med denne protokol, kan relativt rene kulturer af hudfibroblaster opnås. Fibroblasterne har karakteristiske morfologiske træk aflange spindel-lignende celle organer, runde til ovale cellekerner, og fibroblasterne vokser afstemt og i bundter når sammenflydende. Medierne understøtter væksten af fibroblaster, mens andre cellepopulationer f.eks keratinocytter brug for ekstra kosttilskud og vækstfaktorer, eller mitotisk mindre aktive, giver dette for relativt rene fibroblastkulturer. <p class="jove_content...

<table border="1">
<tbody>
 <tr>
 <td>Name of Equipment</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>Dissecting microscope</td>
 <td>Leica</td>
 <td>S6E</td>
 </tr>
 <tr>
 <td>10 cm Tissue Culture Petri dish</td>
 <td>VWR</td>
 <td>25382-166</td>
 </tr>
 <tr>
 <td>6-well Tissue Culture plate </td>
 <td>VWR</td>
 <td>73520-906</td>
 </tr>
 <tr>
 <td>Stainless steel disposable sterile scalpels (2), blade No#15, Miltex</td>
 <td>VWR</td>
 <td>21909-660</td>
 </tr>
 <tr>
 <td>Sterile pointed-tip forceps</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>50 mL Conical tubes</td>
 <td>VWR</td>
 <td>21008-940</td>
 </tr>
 <tr>
 <td>2 mL Serological Pipettes</td>
 <td>VWR</td>
 <td>89130-894</td>
 </tr>
 <tr>
 <td>5 mL Serological Pipettes</td>
 <td>VWR</td>
 <td>89130-896</td>
 </tr>
 <tr>
 <td>10 mL Serological Pipettes</td>
 <td>VWR</td>
 <td>89130-898</td>
 </tr>
 <tr>
 <td>Pasteur Pipettes</td>
 <td>VWR</td>
 <td>14672-380</td>
 </tr>
 </tbody>
</table>
Table A. Table of specific reagents and equipments.
 <table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent</td>
 <td>Company </td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>1X DMEM: High Glucose</td>
 <td>Invitrogen/Gibco</td>
 <td>11960-069</td>
 </tr>
 <tr>
 <td>20% Fetal Bovine Serum</td>
 <td>Invitrogen/Gibco</td>
 <td>26140-079</td>
 </tr>
 <tr>
 <td>1X L-Glutamine</td>
 <td>Invitrogen/Gibco</td>
 <td>25030-164</td>
 </tr>
 <tr>
 <td>1X MEM Non-essential Amino Acids </td>
 <td>Invitrogen/Gibco</td>
 <td>11140-076</td>
 </tr>
 <tr>
 <td>1X Penicillin/Streptomycin</td>
 <td>Invitrogen/Gibco</td>
 <td>15140-163</td>
 </tr>
 </tbody>
 </table>
Table B. Reagents.

skin punch biopsy explant culture for derivation of primary human fibroblasts

Væv og cellelinier afledt fra et individ med sygdommen er ideelle kilder til at studere sygdomsrelaterede cellulære fænotyper. Patient-afledte fibroblaster i denne protokol er med held blevet anvendt i afledningen af induceret pluripotente stamceller til model sygdom 1.. Tidlige passager af disse fibroblaster kan også anvendes til cellebaserede funktionelle assays til undersøgelse af specifikke sygdomstilstande veje, mekanismer 2 og efterfølgende medikamentscreeningsanalyse tilgange. Fordelen ved den præsenterede protokollen over enzymatiske procedurer er 1) reproducerbarheden af teknikken fra små mængder væv afledt fra ældre patienter, fx patienter ramt med Parkinsons sygdom, 2) den teknisk enkel metode igennem mere udfordrende metoder anvender enzymatiske behandlinger, og 3 ) tiden overvejelse: denne protokol tager 15-20 minutter og kan udføres umiddelbart efter biopsi ankomst. Enzymatiske behandlinger kan tage op til 4 timer og har problemer medoverdigestion, reduktion af cellelevedygtighed og efterfølgende vedhæftning af celler, når det ikke håndteres korrekt. Denne protokol beskriver dissektion og fremstilling af en 4 mm menneskehud biopsi til afledning af en fibroblast kultur og har en meget høj succesrate hvilket er vigtigt, når der beskæftiger sig med patient-afledte vævsprøver. I denne kultur, migrere keratinocytter ud af biopsi væv i den første uge efter fremstilling. Fibroblaster vises 7-10 dage efter den første udvækst af keratinocytter. DMEM høj glucose medier suppleret med 20% FBS favoriserer væksten af ​​fibroblaster i keratinocytter og fibroblaster vil overvokse keratinocytter. Efter 2 passager keratinocytter er blevet udvandet ud resulterer i relativt homogene fibroblastkulturer der udtrykker fibroblast markør SERPINH1 (HSP-47). Ved hjælp af denne metode kan 15-20.000.000 fibroblaster udledes i 4-8 uger for celle banking. Huden dissektion tager omkring 15-20 min, celler overvåges derefter en gang om dagenunder mikroskopet, medier og skiftes hver 2-3 dage efter fastgørelse og udvækst af celler.

Keratinocytter vokser ud af biopsi stykker, så snart 48 timer efter dissektion. Første fibroblaster udvækst kan ses omkring en uge efter behandlingen. Når brøndene har nået konfluens, er fibroblaster passeres til to flere passager for at nå tre 150 cm kolber (T150), og celler kryokonserveres. Vi generere med denne metode 15-20.000.000 celler. Fibroblasterne er positive for anti-SERPINH1 (også kendt som HSP-47) (figur 1), som er et kollagen-specifik molekylær chaperone lokaliseret i det endoplas...

Watch this Scientific Journal Video about Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts at JoVE.com

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Fibroblast explant kultur-protokol fra menneskers hud hudbiopsier er en teknisk robust og enkel måde at udlede hudceller inden for 4-8 uger for opsparing af omkring 15-20 millioner celler på et lavt antal passager.

Hud Punch Biopsi Eksplantation Culture for Udledning af primære humane fibroblaster

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