Name: derivation of leptomeninges explant cultures from postmortem human brain donors
Uploaded: 2017-01-21T13:00:00
Description: Watch this Scientific Journal Video about Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors at JoVE.com

Development of this protocol was funded by private donations directed to the Parkinson's Institute Brain Donation Program.

Hjernen donasjon registrering inneholder dokumentasjon av registranten av deres hensikt å donere. Obduksjonen tillatelse til vev gjenfinning er levert av pårørende som er tillatt ved lov. Undersøkelser ved hjelp av innsamlede obduksjons prøvene gjennomgås av Institutional Review Board (IRB) for å sikre samsvar med helseforsikring bærbarhet og Accountability Act (HIPAA) forskrifter. MERK: leptomeninges prøver er innsamlet av hjernen dissector eller nevropatologen under en hjerne diss...

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Denne protokollen beskriver en enkel og robust protokoll for å utlede en meningeal fibroblast kultur fra humane postmortem leptomeninges samlet i forbindelse med en hjerne donasjon. Det er svært få beskrivelser av protokoller for å utlede cellekulturer fra postmortem humant materiale. To studier beskriver hud-avledede fibroblastkulturer 7, 8, 9, beskriver en studie dura prøvene 10, og en annen beskr...

Hjernehinnene består av tre membraner som beskytter hjernen: dura mater, arachnoid og pia mater. Mer nylig har det blitt erkjent at hjernehinnene også spille en viktig rolle i utvikling av hjernen og hjerne homeostase 1. Hjernehinnene blir utledet fra mesenchymale og neural crest-avledede celler og interessant, er det blitt vist at stamceller som befinner seg i hjernehinnene kan gi opphav til neuroner in vitro og in vivo etter transplantasjon to, tre, fire. Hjernehinnene kulturer har også blitt brukt med hell som mater lag, da de har stromal celle-avledede induserende aktivitet for differensiering av embryoniske stamceller i dopaminerge neuroner 5. Videre leptomeninges har potensial til direkte å differensiere til neuroner, astrocytter og oligodendrocytter i henhold iskemiske tilstander 6. For denne protokollen er menneskepostmortem meninges prøver samlet inn fra arachnoid og pia mater, kollektivt kalt leptomeninges, og er anskaffet som en del av en menneskelig hjerne donasjon til forskningsformål. Disseksjon av hjernen blir utført i løpet av 24 timer til død og den leptomeninges prøven legges i kaldt vekstmedier for ytterligere behandling i løpet av de neste 6-8 timer som illustrert her i denne protokollen. Denne protokollen beskriver disseksjon og fremstillingen av humane meninges prøver for utvikling av pasienten primære leptomeninges cellekultur. Vevet blir kuttet i 25-30 stykker av omtrent 3 mm x 3 mm kvadrater. Tre stykker er plassert i hver 6-brønnen gelatin-belagt godt og holdt nede med runde dekkglass. Den hjernehinnene disseksjon tar ca 25-35 min. Den største utfordringen med denne kulturen er sterilitet som hjernen innkjøp, transport, og disseksjon blir vanligvis ikke utført under sterile betingelser. Therefore, er det viktig å supplere kulturmediet med en cocktail av penicillin, streptomycin og amfotericin B og bruke multi-brønn skåler for separat kultur vev stykker. Utvekst av meningeale fibroblaster oppstår vanligvis i løpet av den første uken. Media blir endret hver to til tre dager før cellene er konfluent og cellene blir enzymatisk passeres. De meningeale fibroblaster er oppbevart på 1 million celler per ml / hetteglass i nedfrysing media. Med denne protokollen kan 20-30 millioner meningeale fibroblaster utledes i 6-8 uker for nedfrysing. Nedstrøms anvendelser av disse meningeale fibroblaster er primære kulturer for sykdom forskning, direkte neuronal differensiering eller avledning av induserte pluripotente stamceller fra leptomeninges for forståelse av sykdomsmekanismer og for legemiddelutvikling.

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			<td height="22" style="height:22px;width:459px;">Corning Petri dishes</td>
			<td style="width:187px;">Fisher Scientific</td>
			<td style="width:127px;">351029</td>
			<td style="width:485px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Nunc 6-well plate</td>
			<td>Fisher Scientific</td>
			<td>14-832-11</td>
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		<tr height="21">
			<td height="21" style="height:21px;">15-mm cover slips</td>
			<td>Fisher Scientific</td>
			<td colspan="2">12-545-83 15CIR-1D</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Scalpels, sterile blade, No. 15</td>
			<td>Miltex</td>
			<td>4-415</td>
			<td></td>
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			<td height="21" style="height:21px;">Curved precision tip forceps</td>
			<td>Fisher Scientific</td>
			<td>16-100-122</td>
			<td></td>
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			<td height="21" style="height:21px;">Serological pipettes</td>
			<td>Fisher Scientific</td>
			<td>13-678-11E</td>
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			<td height="21" style="height:21px;">Pasteur pipettes</td>
			<td>Fisher Scientific</td>
			<td>22-230490</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Gelatin</td>
			<td>Sigma</td>
			<td>G1890-100G</td>
			<td></td>
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			<td height="21" style="height:21px;">Phosphate Buffer Solution</td>
			<td>Fisher Scientific</td>
			<td>SH30264.02</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Corning 500 mL filter unit</td>
			<td>Fisher Scientific</td>
			<td>430770</td>
			<td>Combine media components and filter.</td>
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		<tr height="24">
			<td height="24" style="height:24px;">Nunc Cell Culture Treated Flasks with Filter Caps, T175 cm2&#160;</td>
			<td>Thermo Scientific</td>
			<td>178883</td>
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			<td></td>
			<td></td>
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			<td height="22" style="height:22px;">Hyclone DMEM</td>
			<td>Fisher Scientific</td>
			<td>SH30081.02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hyclone FBS</td>
			<td>Fisher Scientific</td>
			<td>SH30910.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM Non-Essential Amino Acids Solution (100x)</td>
			<td>Thermo Fisher</td>
			<td>11140-050</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">GlutaMAX Supplement (100x)</td>
			<td>Thermo Fisher</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Pyruvate (100 mM)</td>
			<td>Thermo Fisher</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermo Fisher</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amphotericin B (Yellow Solution/250 &#181;g/mL)</td>
			<td>Fisher Scientific</td>
			<td>BP264520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bambanker Freeze 120 mL</td>
			<td>Fisher Scientific</td>
			<td>NC9582225</td>
			<td></td>
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		<tr height="22">
			<td height="22" style="height:22px;">Name</td>
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			<td>Catalog Number</td>
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		<tr height="23">
			<td height="23" style="height:23px;">Fibronectin Staining</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">8 well chamber slides&#160;</td>
			<td>Fisher Scientific</td>
			<td>1256518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20% paraformaldehyde&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>15713</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100&#160;</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100% Glycerol&#160;</td>
			<td>BioRad</td>
			<td>9455</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100% normal goat serum&#160;</td>
			<td>Fisher Scientific</td>
			<td>101098-382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Fibronectin antibody [F1]</td>
			<td>Abcam</td>
			<td>ab32419</td>
			<td>1:300 dilution in blocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-SERPINH1</td>
			<td>Sigma</td>
			<td>S5950-200ul</td>
			<td>1:250 dilution in blocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-SOX2</td>
			<td>Millipore</td>
			<td>MAB4343</td>
			<td>1:100 dilution in blocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Nestin</td>
			<td>Millipore</td>
			<td>MAB5326</td>
			<td>1:200 dilution in blocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-TUJ1</td>
			<td>Covance</td>
			<td>MMS-435P</td>
			<td>1:1000 dilution in blocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 anti-rabbit</td>
			<td>Thermo Fisher</td>
			<td>A11029</td>
			<td>1:400 dilution in blocking solution; (green channel; Ex/Em2 495/519 nm)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 555 anti-mouse</td>
			<td>Thermo Fisher</td>
			<td>A21424</td>
			<td>1:400 dilution in blocking solution; (red channel; Ex/Em2 590/617 nm)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33342 stain</td>
			<td>Thermo Fisher</td>
			<td>H3570</td>
			<td>dilute to a final concentration of 1.0 &#956;g/ml; (blue channel; Ex/Em2 358/461 nm)&#160;</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Suppliers are suggestions, similar products from alternative vendors can be used as well.</td>
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</table>

derivation of leptomeninges explant cultures from postmortem human brain donors

Selv om store fremskritt har blitt gjort i den kliniske karakteriseringen av Parkinsons sykdom, flere studier rapporterer at diagnosen av Parkinsons sykdom ikke er patologisk bekreftet i opp til 25% av klinisk diagnostisert Parkinsons sykdom. Derfor vev hentet fra klinisk diagnostiserte pasienter med idiopatisk Parkinsons sykdom kan ha en høy grad av feildiagnostisering; derav in vitro studier fra slike vev for å studere Parkinsons sykdom som en preklinisk modell kan bli forgjeves. Ved å samle post mortem menneskelige leptomeninges med en bekreftet nevropatologiske diagnose av Parkinsons sykdom og preget av nigrostriatal celle tap og intracellulære protein slutninger kalt Lewy-legemer, kan man være sikker på at klinisk observert parkinsonisme ikke er forårsaket av en annen underliggende sykdomsprosess (f.eks tumor, arteriosklerose). Denne protokollen gavets disseksjon og utarbeidelse av post mortem menneskelige leptomeninges for avledning av en meningeal fibroblast kultur. Denne fremgangsmåte er robust og har en høy suksessrate. Utfordringen av kulturen er sterilitet som hjernen innkjøp er vanligvis ikke utføres under sterile forhold. Derfor er det viktig å supplere kulturmediet med en cocktail av penicillin, streptomycin og amfotericin B. Avledning av meningeale fibroblaster fra obduksjonsbekreftede tilfeller med Parkinsons sykdom gir grunnlag for in vitro modellering av Parkinsons sykdom. Meningeale fibroblaster vises 3-9 dager etter prøveopparbeidelse og ca 20-30 millioner celler, kan fryses ned i 6-8 uker. Den meningeal fibroblast kultur er homogen og cellene uttrykker fibronektin, en markør som vanligvis brukes til å identifisere hjernehinnene.

Når leptomeninges behandling protokollen har vært vellykket, er utvekst av meningeale fibroblaster først observert tre til ni dager etter disseksjon, selv om dette kan være avhengig av lengden på post-mortem intervall for hjernen. Figur 1 viser meningeale fibroblast kulturer av fire forskjellige givere. Figur 1A viser en leptomeninges stykke holdt nede av et glass dekkglass (mørk diagonal linje) og fibroblast utvekst rundt vevet fire dager etter be...

Watch this Scientific Journal Video about Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors at JoVE.com

Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors

Den leptomeninges eksplantering kultur protokoll fra menneskelige postmortem hjerne er en teknisk robust og enkel måte å utlede fibronektin-positive meningeale fibroblaster innen 6-8 uker, og fryse ned omtrent 20-30 millioner celler.

Avledning av leptomeninges eksplantering kulturer fra postmortem menneskelige hjerne givere

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Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

Effektiv Utledning av Human nevrale stamceller og nevroner fra pluripotent humane embryonale stamceller med lite molekyl Induksjon

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications ...

Derivation of Glial Restricted Precursors from E13 mice

Utledning av glial Begrensede Forstadier fra E13 mus

This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors ...

Neural Explant Cultures from Xenopus laevis

Neural Explant Cultures from Xenopus laevis

Neural Eksplantasjon kulturer fra<em&gt; Xenopus laevis</em

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Stamfar-avledet Oligodendrocyte Kultur System fra Human fosterets hjerne

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Feeder-free Utledning av Neural Crest stamceller fra menneskelige pluripotent stamceller

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Avledning av voksent menneske fibroblaster og deres direkte konvertering til Utvid Neural stamceller

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

Høyoppløselig Structural Magnetic Resonance Imaging of Human subcortex<I&gt; I Vivo</I&gt; Og Post mortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of ...