Name: a cell culture model of resistance arteries
Uploaded: 2017-09-08T12:00:00
Description: Watch this Scientific Journal Video about A Cell Culture Model of Resistance Arteries at JoVE.com

Dette arbejde blev støttet af National Heart, giver Lung, og Blood Institute R01088554, T32HL007284 og American Heart Association predoctoral fellowship 14PRE20420024. Vi takker især St. Jude cellulære Imaging delt forskning kernen, herunder Randall Wakefield og Linda Horner for elektronmikroskopi billeder, Adam Straub for bistand med repræsentative immunofluorescens billeder og Pooneh Bagher for hende værdifuld feedback på manuskript-protokollen.

1. plating celler for VCCC <ol><li>kultur store 225 cm 2 flasker af EF og SMC under sterile forhold ved 37 ° C, indtil 70-90% sammenflydende. Sikre, at flere EF end SMC bliver brugt; Hvis bruger primære menneskelige EF og SMC, typisk 3 store kolber i EF til 1 stor kolbe af SMC.
	<ol><li>Bruger primærelementer på lavere passager og brug ikke forbi passage 10. Vælg dyrkningsmedier baseret på celler til at være co kulturperler. Primære menneskelige koronar arterie EF, at bruge EBM-2 EF...

<ol>
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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+injury+induces+vascular+smooth+muscle+cell+proliferation+in+highly+localized+regions+of+a+direct+contact+co-culture+system.">Endothelial injury induces vascular smooth muscle cell proliferation in highly localized regions of a direct contact co-culture system.</a> Cell Biochem Biophys. 52 (1), 37-46 (2008).</li><li>Scott-Drechsel, D., 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=A+new+flow+co-culture+system+for+studying+mechanobiology+effects+of+pulse+flow+waves.">A new flow co-culture system for studying mechanobiology effects of pulse flow waves.</a> Cytotechnology. 64 (6), 649-666 (2012).</li><li>van Buul-Wortelboer, M. F., 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=Reconstitution+of+the+vascular+wall+in+vitro.+A+novel+model+to+study+interactions+between+endothelial+and+smooth+muscle+cells.">Reconstitution of the vascular wall in vitro. A novel model to study interactions between endothelial and smooth muscle cells.</a> Exp Cell Res. 162 (1), 151-158 (1986).</li><li>Wilhelm, I., Fazakas, C., Krizbai, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+models+of+the+blood-brain+barrier.">In vitro models of the blood-brain barrier.</a> Acta Neurobiol Exp (Wars). 71 (1), 113-128 (2011).</li><li>Heberlein, K. 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C., 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=Compartmentalized+connexin+43+s-nitrosylation/denitrosylation+regulates+heterocellular+communication+in+the+vessel+wall.">Compartmentalized connexin 43 s-nitrosylation/denitrosylation regulates heterocellular communication in the vessel wall.</a> Arterioscler Thromb Vasc Biol. 31 (2), 399-407 (2011).</li><li>Cucina, A., 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=Vascular+endothelial+growth+factor+increases+the+migration+and+proliferation+of+smooth+muscle+cells+through+the+mediation+of+growth+factors+released+by+endothelial+cells.">Vascular endothelial growth factor increases the migration and proliferation of smooth muscle cells through the mediation of growth factors released by endothelial cells.</a> J Surg Res. 109 (1), 16-23 (2003).</li><li>Heberlein, K. R., 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=Plasminogen+activator+inhibitor-1+regulates+myoendothelial+junction+formation.">Plasminogen activator inhibitor-1 regulates myoendothelial junction formation.</a> Circ Res. 106 (6), 1092-1102 (2010).</li><li>Isakson, B. E., Best, A. K., Duling, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+protein+on+actin+bridges+between+endothelium+and+smooth+muscle+in+arterioles+demonstrates+heterogeneous+connexin+expression+and+phosphorylation.">Incidence of protein on actin bridges between endothelium and smooth muscle in arterioles demonstrates heterogeneous connexin expression and phosphorylation.</a> Am J Physiol Heart Circ Physiol. 294 (6), H2898-H2904 (2008).</li><li>Biwer, L. A., Isakson, B. 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VCCC har en række fordele i form af recapitulating MEJs i vitro, men der er punkter i diskussionen ved fastsættelsen af, hvis VCCC kan udnyttes til specifik anvendelse. For eksempel, hvis forskellige celletyper der skal bruges med denne model, skal det optimeres. Vi har brugt primære menneskelige celler, men det er muligt at isolere cellerne fra kvæg aortas24eller vildtype eller knockout mus, og bruge dem i VCCC1,5,...

I lille diameter modstand arterier adskiller en tynd indre elastisk lamina (IEL) endotel (EF) og glatte muskelceller (SMC). Cellerne kan projektet gennem huller i denne elastiske matrix og gøre direkte cytoplasmatisk forbindelser via gap junction kanaler1,2. Denne unikke struktur kaldes myoendothelial krydset (MEJ). MEJ er en lille (ca 0,5 µm x 0,5 µm, afhængigt af den vaskulære seng), cellulære projektion består overvejende i endothelial celler men kan stamme fra glatte muskulatur samt3,4. En række af efterforskere har vist den enorme kompleksitet signalering netværk, der forekommer selektivt på MEJ, gengivelse sig en særlig vigtig placering for at lette bi-directional signalering mellem endothelium og glatte muskulatur5 ,6,7,8,9,10.
Men en mekanistisk dissektion af signaling veje på MEJ er vanskeligt i en intakt arterie. Fordi MEJ er en cellulær projektion, er det ikke i øjeblikket muligt at isolere en i vivo MEJ fra det vaskulære væg. Af denne grund, blev VCCC model1 udviklet. Vigtigere er, VCCC replikater fysiologiske endotel morfologi11 og polarisering af signaler mellem den apikale og MEJ dele af celle12. Det var denne unikke model, der fremmes den opdagelse, at alpha hæmoglobin er i endothelial cellen polariseret til MEJ. Dette er i modsætning til konventionelt dyrkede endotelceller, som ikke udtrykker alpha hæmoglobin13. For forskere interesseret i mikrovaskulære endothelium, kan det være mere hensigtsmæssigt at bruge endotelceller, der har været kulturperler i VCCC, især hvis hensigten er at dissekere signaling veje, der forekommer i lille diameter modstand arterier.
Ved hjælp af en robust plastindsats der indeholder et filter med lille diameter porer (0.4 µm i diameter) til fælles kultur to forskellige celletyper forhindrer cellerne i overflytning mellem lagene. Det resulterer i en 10 µm afstand mellem celler, som er betydeligt længere end in vivo, men stadig replikater mange MEJs, herunder protein lokalisering og anden messenger signalering1 i vivo karakteristika , 14. Desuden, at VCCC giver mulighed for målretning af celle type-specifikke signaler via tilføjelsen af en agonist og antagonist til den specifikke cellulære rum. For eksempel, lastning EF med BAPTA-AM til Chelat calcium, og stimulere SMC med phenylephrin14. I modsætning til andre beskrivelser af co kultur modeller15,16,17,18,19,20,21giver dette instruktioner på isolere de forskellige fraktioner til mRNA og protein, herunder den særskilte MEJ fraktion inden filter porer. Tilføjelsen af denne teknik til at VCCC giver mulighed for specifikke undersøgelse af ændringer i mRNA lokalisering eller transskription22, protein fosforylering12,23, og protein aktivitet12. Denne artikel vil beskrive plating af EF på toppen af filter og SMC på bunden, selv om det er muligt at kultur to celletyper i forskellige konformationer11,18,24.

<table>
	<tbody>
		<tr>
			<td>24mm diameter, 0.4&#181;m Transwell Polyester membrane filter insert</td>
			<td>Corning</td>
			<td>3450</td>
			<td>This is one 6 well plate of inserts</td>
		</tr>
		<tr>
			<td>225cm2 flask</td>
			<td>Corning</td>
			<td>431082</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plates (without filter inserts)</td>
			<td>Corning</td>
			<td>3506</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary human coronary artery endothelial cells</td>
			<td>Lonza</td>
			<td>CC-2585</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary human coronary artery smooth muscle cells</td>
			<td>Lonza</td>
			<td>CC-2583</td>
			<td></td>
		</tr>
		<tr>
			<td>EBM-2 EC Basal media</td>
			<td>Lonza</td>
			<td>CC-3156</td>
			<td></td>
		</tr>
		<tr>
			<td>EC growth factors (EGM2 MV Single quots)</td>
			<td>Lonza</td>
			<td>CC-4147</td>
			<td>Add to basal media before use</td>
		</tr>
		<tr>
			<td>SmBM SMC basal media</td>
			<td>Lonza</td>
			<td>CC-3181</td>
			<td></td>
		</tr>
		<tr>
			<td>SMC growth factors (SmGM-2 SingleQuot)</td>
			<td>Lonza</td>
			<td>CC-4149</td>
			<td>Add to basal media before use</td>
		</tr>
		<tr>
			<td>0.5% Trypsin-EDTA 10X</td>
			<td>Gibco</td>
			<td>15400-054</td>
			<td>Dilute to 2X with sterile dPBS</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>VWR</td>
			<td>15170-208</td>
			<td></td>
		</tr>
		<tr>
			<td>large petri dishes for SMC Plating (150mm)</td>
			<td>Corning</td>
			<td>353025</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine gelatin</td>
			<td>Sigma</td>
			<td>G-9382</td>
			<td></td>
		</tr>
		<tr>
			<td>Human fibronectin</td>
			<td>Corning</td>
			<td>356008</td>
			<td>5&#181;g/ml of fibronectin and store at -20</td>
		</tr>
		<tr>
			<td>10X Hanks&#39; Buffered salt solution (HBSS)</td>
			<td>Gibco</td>
			<td>14065-056</td>
			<td>Dilute to 1x with sterile water</td>
		</tr>
		<tr>
			<td>10X Dulbecco&#39;s Phosphate buffered saline (dPBS)</td>
			<td>Gibco</td>
			<td>14200-075</td>
			<td>Dilute to 1x with sterile water</td>
		</tr>
		<tr>
			<td>Disposable Curved Scalpel (30mm cutting edge) with handle</td>
			<td>Amazon</td>
			<td>MDS15210</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fisher</td>
			<td>17-467-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell lifter</td>
			<td>Corning</td>
			<td>3008</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>BD Falcon</td>
			<td>353085</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL conical tube</td>
			<td>Corning</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>10mm petri dishes</td>
			<td>Falcon</td>
			<td>35-1008</td>
			<td></td>
		</tr>
		<tr>
			<td>Rneasy mini kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA lysis reagent</td>
			<td>Qiagen</td>
			<td>79306</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>158127</td>
			<td>Make 4% solution with dPBS</td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>Fisher</td>
			<td>04-355-305</td>
			<td></td>
		</tr>
		<tr>
			<td>Cavicide disinfectant</td>
			<td>Fisher</td>
			<td>131000</td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA protein lysis buffer</td>
			<td>Sigma</td>
			<td>R0278</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonic dismembranator</td>
			<td>Fisher</td>
			<td>Model 50</td>
			<td></td>
		</tr>
		<tr>
			<td>1X PBS for harvesting EC/SMC and immunocytochemistry</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td>does not need to be sterile</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Embedding/Sectioning/Staining</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Fisher</td>
			<td>P31-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated tissue processer</td>
			<td>Excelsior</td>
			<td>ES</td>
			<td></td>
		</tr>
		<tr>
			<td>Embedding station + cold plate</td>
			<td>Leica</td>
			<td>HistoCore Arcadia</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Tek Accu-Edge Microtome blade</td>
			<td>VWR</td>
			<td>25608-961 or 25608-964</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Thermo Fisher</td>
			<td>22-230-900</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Thermo Fisher</td>
			<td>12-548-5E</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolong Gold mounting medium</td>
			<td>Thermo Fisher</td>
			<td>P36931</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Other Solutions</td>
			<td></td>
			<td></td>
			<td>**Perform all steps in sterile conditions if using the solution on live cells</td>
		</tr>
		<tr>
			<td>Sterile dPBS</td>
			<td></td>
			<td></td>
			<td>Autoclave water to sterilize. Using the sterile water make DPBS (450mL sterile water: 50mL 10X DPBS), mix and filter sterilize (0.2&#181;m filter). 
			Final concentration: 1X</td>
		</tr>
		<tr>
			<td>sterile HBSS</td>
			<td></td>
			<td></td>
			<td>Autoclave water to sterilize. Using the sterile water make HBSS (450mL sterile water: 50mL 10X HBSS), mix and filter sterilize (0.2&#181;m filter). 
			Final concentration: 1X</td>
		</tr>
		<tr>
			<td>Fibronectin stock solution</td>
			<td></td>
			<td></td>
			<td>Use 50mL of sterile water to fibronectin to make stock of 1mL aliquots, then freeze until needed 
			Final concentration: 100&#181;g/mL</td>
		</tr>
		<tr>
			<td>Fibronectin to coat SMC side of VCCC</td>
			<td></td>
			<td></td>
			<td>thaw one aliquot and add 1mL of fibronectin (100&#181;g/mL) to 19mLs of 1X HBSS to reach a final concentration of 5&#181;g/mL. 
			Final concentration: 5&#181;g/mL</td>
		</tr>
		<tr>
			<td>2x Trypsin EDTA solution</td>
			<td></td>
			<td></td>
			<td>Dilute the 10X 0.5% Trypsin-EDTA to 2X with 1X dPBS (40mL DPBS: 10mL 10X Trypsin) . 
			Final concentration: 2X</td>
		</tr>
		<tr>
			<td>Blocking/antibody solution</td>
			<td></td>
			<td></td>
			<td>(9mL 1X PBS, 25mL Triton X100, 50mg bovine serum albumin, 500mL normal serum)</td>
		</tr>
		<tr>
			<td>Bovine gelatin to coat EC VCCC</td>
			<td></td>
			<td></td>
			<td>Mix 0.5g bovine gelatin into 100mL distilled water. Autoclave prior to first use and store at room temperature. Keep in sterile conditions. 
			Final concentration: 0.50%</td>
		</tr>
		<tr>
			<td>1X PBS for harvesting cells</td>
			<td></td>
			<td></td>
			<td>does not need to be sterile</td>
		</tr>
	</tbody>
</table>

a cell culture model of resistance arteries

Myoendothelial krydset (MEJ), en unik signaling microdomain i lille diameter modstand arterier, udstiller lokalisering af specifikke proteiner og signalering processer, der kan styre vaskulære tone og blodtryk. Da det er en projektion fra enten i endothelial eller glat muskulatur cellen, og på grund af sin lille størrelse (i gennemsnit, et område med ~ 1 µm2), MEJ er svært at studere i isolation. Dog har vi udviklet en celle kultur model kaldet vaskulær Co cellekultur (VCCC), der giver mulighed for in vitro- MEJ dannelse, endotel celle polarisering og dissektion af signalering proteiner og processer i det vaskulære mur af modstand arterier. VCCC har en lang række applikationer og kan tilpasses til forskellige celletyper. Modellen består af to celletyper dyrkes på modsatte sider af et filter med 0,4 µm porer, hvor i vitro MEJs kan danne. Her vi beskrive, hvordan du opretter VCCC via plating af celler og isolation i endothelial, MEJ, og glatte muskulatur fraktioner, som derefter kan bruges til protein isolation eller aktivitet assays. Filter med intakt cellelag kan fastsættes, integrerede og sektioneret immunfluorescent analyse. Vigtigere, er mange af opdagelser fra denne model blevet bekræftet ved hjælp af intakt modstand arterier, understreger dens fysiologiske relevans.

Filter indsætter anvendes til VCCC har små, 0,4 µm huller. Figur 1A, en da ansigt visning af VCCC filter inset, viser porerne, hvor MEJ kan danne i vitro. Skematiske skildrer de glatte muskelceller belagt på den nederste side af filteret én dag før i endothelial celler er belagt på den modsatte side (figur 1B). Når cellerne er forgyldt, kan EF, MEJ og SMC fraktioner være isolerede tre dage senere. Et ek...

Watch this Scientific Journal Video about A Cell Culture Model of Resistance Arteries at JoVE.com

A Cell Culture Model of Resistance Arteries

En celle kultur model af modstand arterier er beskrevet, giver mulighed for dissektion af signalering veje i endothelium, glatte muskelceller, eller mellem endothelium og glat muskulatur (myoendothelial krydset). Selektiv anvendelse af agonister eller protein isolation, elektronmikroskopi eller immunfluorescens kan udnyttes ved hjælp af denne celle kultur model.

En celle kultur Model af modstand arterier

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and ...

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