Name: improved method for the preparation of a human cell-based, contact model of the blood-brain barrier
Uploaded: 2013-11-12T18:45:00
Description: Watch this Scientific Journal Video about Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier at JoVE.com

Denne undersøgelse blev finansieret af tilskud til RLM fra Sundhed og Medical Research Council National of Australia (tilskud # 606.658).

1.. Cell Culture (3-7 dage før BBB Forsamling) 1.1. Primære Menneskelige hjerne mikrovaskulære endotelceller (BEC) BECs blev fremskaffet kommercielt. Cellerne blev fremstillet ved dispase dissociation af normalt humant Hjernebarkvæv og forudsat frosset ved passage 3 (&lt;12 populationsfordoblinger). <ol><li> Undergrunden: Frakke vævs-kultur fartøjer med &quot;Attachment Factor&quot; som pr fabrikantens anvisninger. </li><li> Cell vedligeholdelse...

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Medicinsk forskning i hjernens sygdomme lider meget translationel vanskeligheder. På området for akut iskæmisk slagtilfælde, for eksempel, mange lægemidler, der viste stor løfte i dyremodeller mislykkedes på klinikken 38,39. Årsagerne til disse skuffende resultater er mangfoldige og omfatter utroskab af de prækliniske testsystemer til den menneskelige slagtilfælde scenario og overdrivelse af resultater fra dyreforsøg 21. En strategi for at forbedre den prædiktive værdi af prækliniske ...

BBB er et specialiseret grænseflade mellem det perifere blodcirkulation og det centrale nervesystem, afgørende er ansvarlig for vedligeholdelse af hjernens hæmostase. Det består af særskilte hjerneceller mikrovaskulære endotelceller (BECs), som er funktionelt påvirket af nogle cellulære og acellulære komponenter (nedenfor) for at danne en tæt og dynamisk gateway ind i hjernen. Under fysiologiske betingelser BBB begrænser passagen af ​​blodceller, plasma komponenter og skadelige stoffer, alle potentielt neurotoksiske, ind i hjernen. Sideløbende BBB selektivt udveksler vigtige ioner og næringsstoffer (glukose og aminosyrer) og metaboliske affaldsprodukter mellem hjernen og cirkulation til præcist at opretholde hjernen miljø 1,2. I de seneste år er det blevet klart, at svigt af BBB forekommer i en række kroniske hjernen patologier, såsom neurodegenerative eller inflammatoriske sygdomme (fx Alzheimers sygdom og flee sklerose, henholdsvis) 3, samt i akutte tilstande som iskæmisk slagtilfælde 4. De unikke BBB egenskaber af hjernens endotelceller (BECs) er stort set fremkaldt af deres cerebral miljø 5, og især af astrocytter 6,7. Der er en voksende forståelse for, at andre celletyper, såsom pericytter 8, neuroner og mikroglia 1,3 samt basalmembranen 9 støtter BECs og sammen danner en funktionel enhed benævnt &quot;neurovaskulær enhed&quot; (NVU), som samtidig par neuronal metaboliske krav til deres leverer kapillærer 10. Inddragelsen af BBB i patologiske situationer ligger bag mange forsøg på at udvikle in vitro BBB modeller til at hjælpe med BBB-relateret forskning 11,12. Disse modeller har til formål at efterligne så tæt som muligt in vivo BBB egenskaber ifølge NVU princip. In vitro </em&gt; BBB modeller generelt er afhængige af et monolag af stram-junction-dannende BEC (primært fra kvæg 13, human 14, rotte 15, mus 16, og svin 17,18 oprindelser), dyrket på en porøs membran sammen med understøttende astrocytter (udstrakt revideret af Deli et al. 2005 11). Astrocytter kan dyrkes i ikke-kontakt betingelser på bunden af en vævskultur godt adskilt fra BECs (dyrket på den øvre overflade af membranen) af dyrkningsmediet endnu kommunikere med BECs via opløselige faktorer 16. I mere avancerede modeller, som bedre ligner den anatomiske struktur af BBB in vivo, er astrocytter holdes i kontakt betingelser og dyrkes direkte på den modsatte side af membranen i umiddelbar nærhed af BECs 13,15,17 (fig. 1). Denne konfiguration gør det muligt for fysisk kontakt mellem BEC og astrocytter, etableres, når astrocytter projektderes processer gennem den porøse membran. Vigtigere er det, for en ægte kontakt til forekomme porerne skal være ≥ 1 um i diameter, da astrocytic endelige fødder ikke kan passere gennem mindre pore størrelser (dvs. 0,4 um) 14,15. Især er kontakten BBB systemer vist i nogle undersøgelser at være overlegen i forhold til deres ikke-kontakt modparter for deres trans-endotel elektrisk modstand (TEER) og endotelpermeabilitet værdier af forskellige sporstoffer 13,17,18. En yderligere dimension af mediestrømmen nylig blev tilsat i en række in vitro BBB modeller at anvende forskydningskræfter til endotelet tættere simulering af hjernen vaskulaturen 12,19. En teknisk forhindring at overvinde, når du genererer en kontakt BBB model er udsåning af astrocytter mod tyngdekraften på den abluminale overflade af den porøse membran. Tidligere protokoller 13,20, hvor astrocytter var simpelthen podet i en dråbe medier oven på en ikonverteres insert, tillod kun kort seeding gange (dvs. 10 min 13 eller 2 hr 20), som blev fundet i vores hænder til at være utilstrækkelig for korrekt fastgørelse celle. Ved hjælp af denne grundlæggende metode, kræver en længere vedhæftet astrocyte periode konstant overvågning af skærene ved hyppig åbning af kuvøsen (forårsager udsving i temperatur, pH og fugtighed), og er også tilbøjelig til ujævn cellepodning skyldes lækage af medier gennem porerne, især hvis porerne er større end 1 um er ansat. Her beskriver vi en generel protokol for udarbejdelse af en kontakt BBB model. Vores procedure omfatter en alternativ metode til cellepodning på omvendte skær, som omhandler de ovennævnte begrænsninger. Metoden tillader uforstyrret vedhæftning af astrocytter på abluminale membranoverfladen i en ækvilibreret inkubator i en længere periode. Som et resultat heraf er en ensartet udsåning af astrocytter opnået som øger barriere kvalitetog minimerer basale variationer permeabilitet mellem skær. Som det er vigtigt for menneske-relevant forskning 21 anvendelsen af humane celler, vi derudover demonstrere i denne artikel den specifikke udnyttelse af en ny kombination af primære humane BEC og udødeliggjort menneskelige astrocytter for etablering af en kontakt human BBB model med en high throughput screening kapacitet . Da visning af celler på porøse membraner kan være svært, har vi også detalje farvningsteknikker som kan hjælpe i bestemmelsen af ​​sammenløb og cellemorfologi på porøse membraner. Endelig vil vi eksemplificere, hvordan vores menneskelige BBB-modellen kan bruges til at undersøge effekten af ​​vævstype-plasminogen-aktivator (t-PA) - en blodprop sprænge enzym, der fungerer som en eneste behandlingsmulighed for akut iskæmisk slagtilfælde - på BBB.

<table border="0" cellpadding="0" cellspacing="0" width="863">
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		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Attachment Factor</td>
			<td style="width:193px;">Cell-Systems Corporation</td>
			<td style="width:171px;">4Z0-210</td>
			<td style="width:243px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Brain microvascular endothelial cells (BECs), primary, human</td>
			<td style="width:193px;">Cell-Systems Corporation</td>
			<td style="width:171px;">ACBRI 376</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Complete medium</td>
			<td style="width:193px;">Cell-Systems Corporation</td>
			<td style="width:171px;">4Z0-500</td>
			<td style="width:243px;">Supplemented with CSC JetFuel</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Complete serum-free medium</td>
			<td style="width:193px;">Cell-Systems Corporation</td>
			<td style="width:171px;">SF-4Z0-500</td>
			<td style="width:243px;">Supplemented with CSC RocketFue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">DMEM/F-12 with 15 mM HEPES</td>
			<td style="width:193px;">Life Technologies</td>
			<td style="width:171px;">11330-032</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Endothelial cells growth supplement (from bovine origin)</td>
			<td style="width:193px;">Sigma-Aldrich</td>
			<td style="width:171px;">E2759-15MG</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">External silicone tubing</td>
			<td style="width:193px;">Watson-Marlow</td>
			<td style="width:171px;">913.A080.016</td>
			<td style="width:243px;">Pumpsil brand, 8 mm internal diameter, 1.6 mm wall</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Foetal calf serum</td>
			<td style="width:193px;">Lonza</td>
			<td style="width:171px;">14-501F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Gentamycin sulfate</td>
			<td style="width:193px;">Life Technologies</td>
			<td style="width:171px;">15750-060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Heparin sodium</td>
			<td style="width:193px;">Pfizer</td>
			<td style="width:171px;"></td>
			<td style="width:243px;">1,000 U/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Hexamethyldisilazane (HMDS)</td>
			<td style="width:193px;">Sigma-Aldrich</td>
			<td style="width:171px;">H4875</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Human brain microvascular endothelial cells</td>
			<td style="width:193px;">Cell-Systems Corporation</td>
			<td style="width:171px;">ACBRI 376</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Internal silicone tubing</td>
			<td style="width:193px;">Watson-Marlow</td>
			<td style="width:171px;">913.A032.016</td>
			<td style="width:243px;">Pumpsil brand, 3.2 mm internal diameter, 1.6 mm wall</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">L-Glutamine</td>
			<td style="width:193px;">Life Technologies</td>
			<td style="width:171px;">25030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Mayer&#8217;s hematoxylin solution</td>
			<td style="width:193px;">Amber Scientific</td>
			<td style="width:171px;">MH</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Minimum essential medium with Earle&#8217;s balanced salt solution</td>
			<td style="width:193px;">HyClone Laboratories</td>
			<td style="width:171px;">SH30244.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Penicillin/Streptomycin</td>
			<td style="width:193px;">Life Technologies</td>
			<td style="width:171px;">15140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Rat collagen I</td>
			<td style="width:193px;">Trevigen</td>
			<td style="width:171px;">3440-100-01</td>
			<td style="width:243px;">Cultrex brand</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:257px;">Tissue culture inserts</td>
			<td style="width:193px;">Corning Life Sciences</td>
			<td style="width:171px;">3472</td>
			<td style="width:243px;">Transwell brand, 6.5 mm in diameter, with polyester porous membrane, 3 &#181;m pore-size</td>
		</tr>
	</tbody>
</table>

improved method for the preparation of a human cell-based, contact model of the blood-brain barrier

Blod-hjerne-barrieren (BBB) ​​omfatter uigennemtrængelige, men fleksible hjernen kapillærer, som stramt styrer hjernen miljø. Svigt i BBB er underforstået i ætiologien af mange hjernen patologier, skaber et behov for udvikling af human in vitro BBB modeller til at hjælpe med klinisk relevante forskning. Blandt de mange BBB modeller hidtil beskrevet, en statisk (uden flow) kontakte BBB-modellen, hvor astrocytter og hjerne endotelceller (BECs) er samdyrket på de modstående sider af en porøs membran, dukket op som en forenklet endnu autentisk systemet til at simulere BBB med højkapacitetsscreening kapacitet. Ikke desto mindre er den generation af en sådan model giver nogle tekniske udfordringer. Her beskriver vi en protokol for forberedelse af en kontaktperson human BBB model udnytte en ny kombination af primære humane BEC og udødeliggjort menneskelige astrocytter. Konkret har vi detalje en innovativ metode til cellepodning på omvendte indstik samt specificere isætningt farvningsteknikker og eksemplificere, hvordan vi bruger vores model for BBB-relateret forskning.

For at etablere et menneske, kontakt BBB model, vi havde til at dyrke SVGs og BEC på porøse membraner med en 3 m pore-størrelse, der er vist til at tillade passage af astrocyt endelige fødder i kontakt med endothelceller 14,15,27,28. En skematisk repræsentation af fuldstændig kontakt model er illustreret i figur 1 (venstre billede). Den største tekniske udfordring præsenteres af en kontakt, er behovet for frø astrocytter mod tyngdekraften på den abluminale overflade af membranen. De...

Watch this Scientific Journal Video about Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier at JoVE.com

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

Etablering af humane modeller af blod-hjerne-barrieren (BBB) ​​kan gavne forskning i hjernesygdomme forbundet med BBB fiasko. Vi beskriver her en forbedret teknik til fremstilling af en kontakt BBB model, som tillader coculturing menneskelige astrocytter og hjerne endotelceller på de modstående sider af en porøs membran.

Forbedret metode til forberedelse af en menneskelig celle-baseret, Contact Model af blod-hjerne barrieren

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

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In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

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In vitro gennemtrængning af FITC-loaded Ferritins tværs af en rotte blodhjernebarrieren: en model til at studere den Levering af Nanoformulated molekyler

Brain microvascular endothelial cells, supported by pericytes and astrocytes endfeet, are responsible for the low permeation of large hydrosoluble drugs ...

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Lucifer Yellow-en robust Paracellulær permeabilitet markør i en celle model af humant blod-hjerne barrieren

The blood-brain barrier BBB consists of endothelial cells that form a barrier between the systemic circulation and the brain to prevent the exchange of ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

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Disruption of the Blood-Spinal Cord Barrier Using Low-Intensity Focused Ultrasound in a Rat Model

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