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.

registrering Hjernen donation omfatter dokumentation af registranten af ​​deres hensigt til at donere. Obduktionen tilladelse til væv hentning leveres af de pårørende som tilladt ved lov. Forskning undersøgelser med anvendelse af indsamlede autopsiprøver gennemgås af den institutionelle Review Board (IRB) for at sikre overholdelse af Health Insurance Mobilitet og Accountability Act (HIPAA) regler. BEMÆRK: leptomeninges prøverne er indsamlet af hjernen dissektoren eller neuropatho...

<ol>
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Denne protokol beskriver en enkel og robust protokol til at udlede en meningeal fibroblast kultur fra humane postmortem leptomeninges indsamlet i forbindelse med en hjerne donation. Der er meget få beskrivelser af protokoller at udlede cellekulturer fra postmortem humant materiale. To studier beskriver hudafledte fibroblastkulturer 7, 8, 9, en undersøgelse beskriver dura prøver 10, og en anden beskriv...

Meninges består af tre membraner, der beskytter hjernen: dura mater, arachnoid og pia mater. For nylig er det blevet erkendt, at meninges også spille en vigtig rolle i udviklingen af hjernen og hjerne homeostase 1. Meninges er afledt af mesenchymale og crista neuralis-afledte celler og interessant er det blevet vist, at progenitorceller bopæl i meninges kan give anledning til neuroner in vitro og efter transplantation in vivo 2, 3, 4. Meninges kulturer er også med succes anvendt som fødelag, da de besidder stromal celleafledt inducerende aktivitet for differentiering af embryonale stamceller i dopaminerge neuroner 5. Desuden leptomeninges har potentiale til direkte at differentiere til neuroner, astrocytter og oligodendrocytter under iskæmiske tilstande 6. Til denne protokol, er humane postmortem meninges prøver indsamlet fra arachnoid og pia mater, kollektivt kaldet leptomeninges, og er indkøbt som en del af en menneskelig hjerne donation til forskningsformål. Dissektion af hjernen er udført inden for 24 timer fra døden og leptomeninges prøven anbringes i koldt vækstmedier til videre forarbejdning i næste 6-8 timer som illustreret her i denne protokol. Denne protokol beskriver dissektion og fremstilling af humane meninges prøver til udviklingen af ​​patientens primære leptomeninges cellekultur. Vævet skæres i 25-30 stykker af ca. 3 mm x 3 mm kvadrater. Tre stykker anbringes i hver 6-brønds gelatineovertrukket godt og holdes nede med runde dækglas. Den meninges dissektion tager omkring 25-35 min. Den største udfordring med denne kultur er sterilitet som hjernen udtagning, transport og dissektion generelt ikke udføres under sterile betingelser. Therefore, er det vigtigt at supplere dyrkningsmediet med en cocktail af penicillin, streptomycin og amphotericin B og anvende multi-brønds skåle til hver kultur vævsstykker. Udvækst af meningeal fibroblaster sker normalt inden for den første uge. Mediet skiftes hver to til tre dage, indtil cellerne er sammenflydende og cellerne enzymatisk passage. De meningeale fibroblaster kryokonserveres ved 1 million celler pr ml / hætteglas i kryopræservering medier. Med denne protokol, kan 20-30 millioner meningeal fibroblaster udledes i 6-8 uger for nedfrysning. Downstream anvendelser af disse meningeal fibroblaster er primære kulturer til sygdom forskning, direkte neuronal differentiering eller afledning af inducerede pluripotente stamceller fra leptomeninges for forståelse af sygdomsmekanismer og for lægemiddeludvikling.

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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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nunc 6-well plate</td>
			<td>Fisher Scientific</td>
			<td>14-832-11</td>
			<td></td>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Curved precision tip forceps</td>
			<td>Fisher Scientific</td>
			<td>16-100-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Serological pipettes</td>
			<td>Fisher Scientific</td>
			<td>13-678-11E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pasteur pipettes</td>
			<td>Fisher Scientific</td>
			<td>22-230490</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gelatin</td>
			<td>Sigma</td>
			<td>G1890-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffer Solution</td>
			<td>Fisher Scientific</td>
			<td>SH30264.02</td>
			<td></td>
		</tr>
		<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>
			<td></td>
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		<tr height="22">
			<td height="22" style="height:22px;">Name</td>
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			<td height="23" style="height:23px;">Growth Media</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr height="22">
			<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>
		</tr>
		<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>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</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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derivation of leptomeninges explant cultures from postmortem human brain donors

Selv om der er gjort store fremskridt i den kliniske karakterisering af Parkinsons sygdom, rapporterer flere undersøgelser, at diagnosticering af Parkinsons sygdom ikke er patologisk bekræftet hos op til 25% af klinisk diagnosticeret Parkinsons sygdom. Derfor væv opsamlet fra klinisk diagnosticerede patienter med idiopatisk Parkinsons sygdom kan have en høj fejldiagnose; dermed in vitro-undersøgelser fra sådanne væv for at studere Parkinsons sygdom som en præklinisk model kan blive forgæves. Ved at samle postmortem human leptomeninges med en bekræftet neuropatologisk diagnose af Parkinsons sygdom og karakteriseret ved nigrostriatale celletab og intracellulære protein inklusioner kaldet Lewy-legemer, kan man være sikker på, at klinisk observeret parkinsonisme ikke er forårsaget af en anden underliggende sygdomsproces (f.eks tumor, arteriosklerose). Denne protokol Presents dissektion og forberedelse af postmortem menneskelige leptomeninges til afledning af en meningeal fibroblast kultur. Denne procedure er robust og har en høj succesrate. Udfordringen af ​​kulturen er sterilitet som hjernen indkøb generelt ikke udføres under sterile forhold. Derfor er det vigtigt at supplere dyrkningsmediet med en cocktail af penicillin, streptomycin og amphotericin B. Udledningen af meningitis fibroblaster fra autopsi-bekræftede tilfælde med Parkinsons sygdom giver grundlaget for in vitro-modellering af Parkinsons sygdom. Meningeal fibroblaster vises 3-9 dage efter prøveforberedelse og omkring 20-30 millioner celler kan kryopræserveres i 6-8 uger. Den meningeal fibroblast kultur er homogen og cellerne udtrykker fibronectin, et almindeligt anvendt markør til at identificere meninges.

Når leptomeninges behandlingsprotokol har været en succes, er udvækst af meningitis fibroblaster først observeret tre til ni dage efter dissektion, selv om dette kan afhænge af længden af ​​intervallet post-mortem for hjernen. Figur 1 viser meningeale fibroblastkulturer af fire forskellige donorer. Figur 1A viser en leptomeninges stykke holdt nede af et glas dækglas (mørk diagonal linje) og fibroblast udvækst omkring væv fire dage efter beh...

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 eksplantat kultur protokol fra human postmortem hjerne er en teknisk robust og enkel måde at udlede fibronektin-positive meningitis fibroblaster inden for 6-8 uger, og Cryopreservering ca. 20-30 millioner celler.

Udledning af leptomeninges eksplantatkulturer fra Postmortem menneskehjerne donorer

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Effektiv derivering af humane Neuronal progenitorer og neuroner fra Pluripotente humane embryonale stamceller med småmolekyle Induktion

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

Udledning af Glia begrænsede prækursorer 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

Neurale eksplantatkulturer 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

Stamfader-afledt oligodendrocyt Culture System fra human føtal 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-fri Udledning af neurale Crest stamceller fra menneskelige pluripotente 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

Udledning af Voksen humane fibroblaster og deres direkte konvertering til udvides Neural progenitorceller

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

Høj opløsning Strukturel Magnetic Resonance Imaging af Human subcortex<I&gt; In vivo</I&gt; Og Postmortem

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 ...