Name: real-time monitoring of human glioma cell migration on dorsal root ganglion axon-oligodendrocyte co-cultures
Uploaded: 2019-12-13T12:00:00
Description: Watch this Scientific Journal Video about Real-Time Monitoring of Human Glioma Cell Migration on Dorsal Root Ganglion Axon-Oligodendrocyte Co-Cultures at JoVE.com

Dette arbejde blev støttet af interne fonde af Department of neurosurgery, Brown University til N.T.

Protokollerne for indsamling, isolering og formering af patient-afledte humane gliom celler blev godkendt af IRB-Komitéen for Rhode Island Hospital. Alle dyr blev vedligeholdt i henhold til NIH guide til pleje og brug af forsøgsdyr. Alle protokoller om anvendelse af dyr blev godkendt af det institutionelle udvalg for dyrepasning og-anvendelse på Rhode Island Hospital.
1. medier og buffer præparater
<ol><li>Forbered 50 mL Neuro Sphere medier: 1x neuronal basal medium w/o vitamin A, 1x ser...

<ol>
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S., Lannutti, J. J., Viapiano, M. S., Sarkar, A., Winter, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+3D+biomimetic+models+to+understand+the+behavior+of+glioblastoma+multiforme+cells.">Toward 3D biomimetic models to understand the behavior of glioblastoma multiforme cells.</a> Tissue Engineering Part B Reviews. 20 (4), 314-327 (2014).</li><li>Windebank, A. J., Wood, P., Bunge, R. P., Dyck, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelination+determines+the+caliber+of+dorsal+root+ganglion+neurons+in+culture.">Myelination determines the caliber of dorsal root ganglion neurons in culture.</a> Journal of Neuroscience. 5 (6), 1563-1569 (1985).</li><li>Wood, P. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contact-dependent+demyelination+by+Mycobacterium+leprae+in+the+absence+of+immune+cells.">Contact-dependent demyelination by Mycobacterium leprae in the absence of immune cells.</a> Science. 296 (5569), 927-931 (2002).</li><li>Tapinos, N., Ohnishi, M., Rambukkana, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ErbB2+receptor+tyrosine+kinase+signaling+mediates+early+demyelination+induced+by+leprosy+bacilli.">ErbB2 receptor tyrosine kinase signaling mediates early demyelination induced by leprosy bacilli.</a> Nature Medicine. 12 (8), 961-966 (2006).</li><li>Chan, J. 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=NGF+controls+axonal+receptivity+to+myelination+by+Schwann+cells+or+oligodendrocytes.">NGF controls axonal receptivity to myelination by Schwann cells or oligodendrocytes.</a> Neuron. 43 (2), 183-191 (2004).</li><li>Dugas, J. C., Tai, Y. C., Speed, T. P., Ngai, J., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+genomic+analysis+of+oligodendrocyte+differentiation.">Functional genomic analysis of oligodendrocyte differentiation.</a> Journal of Neuroscience. 26 (43), 10967-10983 (2006).</li><li>Ruffini, F., Arbour, N., Blain, M., Olivier, A., Antel, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinctive+properties+of+human+adult+brain-derived+myelin+progenitor+cells.">Distinctive properties of human adult brain-derived myelin progenitor cells.</a> American Journal of Pathology. 165 (6), 2167-2175 (2004).</li><li>Zepecki, J. P., Snyder, K. M., Moreno, M. M., Fajardo, E., Fiser, A., Ness, J., Sarkar, A., Toms, S. A., Tapinos, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+human+glioma+cell+migration,+tumor+growth,+and+stemness+gene+expression+using+a+Lck+targeted+inhibitor.">Regulation of human glioma cell migration, tumor growth, and stemness gene expression using a Lck targeted inhibitor.</a> Oncogene. 38, 1734-1750 (2018).</li><li>Chen, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Boyden+chamber+assay.">Boyden chamber assay.</a> Methods in Molecular Bioliogy. 294, 15-22 (2005).</li><li>Merz, 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=Organotypic+slice+cultures+of+human+glioblastoma+reveal+different+susceptibilities+to+treatments.">Organotypic slice cultures of human glioblastoma reveal different susceptibilities to treatments.</a> Neuro-Oncology. 15 (6), 670-681 (2013).</li><li>Singh, S. K., 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=Identification+of+human+brain+tumour+initiating+cells.">Identification of human brain tumour initiating cells.</a> Nature. 432 (7015), 396-401 (2004).</li><li>Aubert, M., Badoual, M., Christov, C., Grammaticos, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+glioma+cell+migration+on+collagen+and+astrocytes.">A model for glioma cell migration on collagen and astrocytes.</a> Journal of the Royal Society Interface. 5 (18), 75-83 (2008).</li><li>Jia, W., 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=Effects+of+three-dimensional+collagen+scaffolds+on+the+expression+profiles+and+biological+functions+of+glioma+cells.">Effects of three-dimensional collagen scaffolds on the expression profiles and biological functions of glioma cells.</a> International Journal of Oncology. 52 (6), 1787-1800 (2018).</li><li>Kaphle, P., Li, Y., Yao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanical+and+pharmacological+regulation+of+glioblastoma+cell+migration+in+3D+matrices.">The mechanical and pharmacological regulation of glioblastoma cell migration in 3D matrices.</a> Journal of Cellular Physiology. 234 (4), 3948-3960 (2019).</li><li>Gritsenko, P., Leenders, W., Friedl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recapitulating+in+vivo-like+plasticity+of+glioma+cell+invasion+along+blood+vessels+and+in+astrocyte-rich+stroma.">Recapitulating in vivo-like plasticity of glioma cell invasion along blood vessels and in astrocyte-rich stroma.</a> Histochemistry and Cell Biology. 148 (4), 395-406 (2017).</li><li>Gritsenko, P. G., Friedl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptive+adhesion+systems+mediate+glioma+cell+invasion+in+complex+environments.">Adaptive adhesion systems mediate glioma cell invasion in complex environments.</a> Journal of Cell Science. 131 (15), (2018).</li><li>Kaur, H., 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=Cadherin-11,+a+marker+of+the+mesenchymal+phenotype,+regulates+glioblastoma+cell+migration+and+survival+in+vivo.">Cadherin-11, a marker of the mesenchymal phenotype, regulates glioblastoma cell migration and survival in vivo.</a> Molecular Cancer Research. 10 (3), 293-304 (2012).</li><li>Rao, S. S., 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=Mimicking+white+matter+tract+topography+using+core-shell+electrospun+nanofibers+to+examine+migration+of+malignant+brain+tumors.">Mimicking white matter tract topography using core-shell electrospun nanofibers to examine migration of malignant brain tumors.</a> Biomaterials. 34 (21), 5181-5190 (2013).</li><li>Huang, Y., 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=Three-dimensional+hydrogel+is+suitable+for+targeted+investigation+of+amoeboid+migration+of+glioma+cells.">Three-dimensional hydrogel is suitable for targeted investigation of amoeboid migration of glioma cells.</a> Molecular Medicine Reports. 17 (1), 250-256 (2018).</li></ol>

Migrationsundersøgelser for hGCs kan udføres ved hjælp af BoyDen kammer systemer eller ridse assays. Men, mens disse eksperimenter undlader at give nogen oplysninger om samspillet mellem tumorceller med andre omgivende væv, det nuværende system kan rekapitulere GC interaktioner med myelinerede og ikke-myelerede fibre. Endvidere, at studere tumordannelse og slutpunkt migration, organotypiske skive kulturer af gnaver hjernen eller in vivo implantation af gliom celler i gnaver hjernen eller flanke har tidligere været ...

Glioblastoma er en af de mest aggressive og dødbringende tumorer i den menneskelige hjerne. Den nuværende standard for pleje omfatter kirurgisk resektion af tumoren efterfulgt af stråling1 plus samtidig og adjuverende administration af temozolomid2. Selv med denne multi-terapeutiske tilgang, tumor gentagelse er uundgåelig3. Dette skyldes til dels den omfattende migrerende karakter af tumorceller, som invaderer hjernen parenkym skabe flere finger-lignende fremskrivninger i hjernen4 , der gør fuldstændig resektion usandsynligt.
I de seneste år, er det blevet klart, at aggressivitet af glioblastoma skyldes, til dels, tilstedeværelsen af en population af kræft stamceller i tumormassen5,6, som udviser højt migrerende potentiale7,8, resistens over for kemoterapi og stråling9,10 og evnen til at danne sekundære tumorer11. GSCs er i stand til at rekapitulere oprindelige polyklonale tumorer, når xenografted til Nude mus5.
På trods af den rigdom af viden om Glioblastomas genetiske baggrund, er undersøgelser af gliom Cell (GC) migration i øjeblikket hæmmet af mangel på effektive in vitro-eller in vivo-migrations modeller. Især mens gliom celle-axonal interaktioner moduleret af cellulære og miljømæssige faktorer er en kernekomponent i gliom invasion, til vores viden er der i øjeblikket ingen eksperimentel system med evnen til at modellere disse interaktioner12,13,14. For at afhjælpe denne mangel udviklede vi et ex vivo-kultur system af primære Hgc'er, der er co-dyrket med rensede DRG Axon-oligodendrocytter, som resulterer i forhøjet ekspression af differentierede tumor markører samt omfattende migration og interaktion af hGCs med myelinerede og ikke-myelerede fibre. Denne ex vivo platform, på grund af sin opdelte layout, er egnet til at teste virkningerne af nye Therapeutics på hGC migration mønstre.,

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 mm Suspension Culture Dish</td>
			<td>Corning</td>
			<td>430591</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5S NGF</td>
			<td>ENVIGO</td>
			<td>B.5025</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm Suspension Culture Dish</td>
			<td>Corning</td>
			<td>430589</td>
			<td></td>
		</tr>
		<tr>
			<td>ACK Lysing Buffer</td>
			<td>Thermo Fisher</td>
			<td>A1049201</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Hydroxide Solution</td>
			<td>Fisher Scientific</td>
			<td>A669-500</td>
			<td>Concentrated</td>
		</tr>
		<tr>
			<td>Animal-Free Recombinant Human EGF</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal-Free Recombinant Human FGF-basic (154 a.a.)</td>
			<td>Peprotech</td>
			<td>AF-100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-A2B5 MicroBeads, human, mouse, rat</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-392</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (100X)</td>
			<td>Thermo Fisher</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>AutoMACS Rinsing Solution (PBS, pH 7.2)</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-222</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Thermo Fisher</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement, minus vitamin A</td>
			<td>Thermo Fisher</td>
			<td>12587001</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacteriological Plate</td>
			<td>BD Falcon</td>
			<td>351029</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin</td>
			<td>Sigma</td>
			<td>B4639</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>Campenot Chamber</td>
			<td>Tyler Research</td>
			<td>CAMP-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Dish</td>
			<td>Corning</td>
			<td>430165</td>
			<td>35mm X 10mm</td>
		</tr>
		<tr>
			<td>Cell Strainer</td>
			<td>BD Falcon</td>
			<td>352350</td>
			<td>70 uM, Nylon</td>
		</tr>
		<tr>
			<td>Cell Strainer</td>
			<td>BD Falcon</td>
			<td>352340</td>
			<td>30 uM, Nylon</td>
		</tr>
		<tr>
			<td>Collagenase/Dispase</td>
			<td>Roche</td>
			<td>11097113001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cultrex Rat Collagen I</td>
			<td>Trevigen</td>
			<td>3440-100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Sigma</td>
			<td>G5146</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher</td>
			<td>10313021</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma</td>
			<td>D7291</td>
			<td></td>
		</tr>
		<tr>
			<td>Dow Corning High-Vacuum Grease</td>
			<td>Fisher Scientific</td>
			<td>14-635-5D</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Roboz</td>
			<td>RS-5045</td>
			<td></td>
		</tr>
		<tr>
			<td>E16 Timed Pregnant Sprague Dawley Rat</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EBSS</td>
			<td>Sigma</td>
			<td>E7510</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Hyclone</td>
			<td>SH30070.02</td>
			<td></td>
		</tr>
		<tr>
			<td>FUDR</td>
			<td>Sigma</td>
			<td>F0503</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Thermo Fisher</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Ham&#39;s F-12 Nutrient Mix</td>
			<td>Thermo Fisher</td>
			<td>11765054</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Thermo Fisher</td>
			<td>14175095</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemostatic Forceps</td>
			<td>Roboz</td>
			<td>RS-7035</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin Sodium Salt, 0.2% in PBS</td>
			<td>Stem Cell Technologies</td>
			<td>07980</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypodermic Needle, 18G</td>
			<td>BD</td>
			<td>511097</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium G</td>
			<td>Thermo Fisher</td>
			<td>41400045</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine</td>
			<td>Sigma</td>
			<td>C7477</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Thermo Fisher</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz&#39;s L-15 Medium</td>
			<td>Thermo Fisher</td>
			<td>11415064</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS BSA Stock Solution</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-376</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS MultiStand</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM</td>
			<td>Thermo Fisher</td>
			<td>1190081</td>
			<td></td>
		</tr>
		<tr>
			<td>Mg2SO4</td>
			<td>Sigma</td>
			<td>M2643</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniMACS Separator</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-102</td>
			<td></td>
		</tr>
		<tr>
			<td>MS Columns plus tubes</td>
			<td>Miltenyi Biotec</td>
			<td>130-041-301</td>
			<td></td>
		</tr>
		<tr>
			<td>NAC</td>
			<td>Sigma</td>
			<td>A8199</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>Thermo Fisher</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal-A Medium</td>
			<td>Thermo Fisher</td>
			<td>10888022</td>
			<td></td>
		</tr>
		<tr>
			<td>Ordinary forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>P2 Sprague Dawley Rat Pups</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Worthington</td>
			<td>LS003126</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher</td>
			<td>15140148</td>
			<td></td>
		</tr>
		<tr>
			<td>Pin Rake</td>
			<td>Tyler Research</td>
			<td>CAMP-PR</td>
			<td></td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P8783</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro Accutase Cell Dissociation Reagent</td>
			<td>Thermo Fisher</td>
			<td>A1110501</td>
			<td></td>
		</tr>
		<tr>
			<td>Syrine Grease Applicator</td>
			<td>Tyler Research</td>
			<td>CAMP-GLSS</td>
			<td></td>
		</tr>
		<tr>
			<td>Transferrin</td>
			<td>Sigma</td>
			<td>T2036</td>
			<td></td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Sigma</td>
			<td>U3003</td>
			<td></td>
		</tr>
	</tbody>
</table>

real-time monitoring of human glioma cell migration on dorsal root ganglion axon-oligodendrocyte co-cultures

Glioblastoma er en af de mest aggressive menneskelige kræftformer på grund af omfattende cellulære heterogenitet og migrations egenskaber af hGCs. For bedre at forstå de molekylære mekanismer underliggende gliom celle migration, en evne til at studere samspillet mellem hgcs og axoner i tumor mikromiljø er afgørende. For at modellere denne cellulære interaktion, udviklede vi et blandet kultur system bestående af hgcs og dorsale root basale ganglier (DRG) Axon-oligodendrocyte Co-kulturer. DRG-kulturerne blev udvalgt, fordi de kan isoleres effektivt og kan danne de lange, omfattende fremskrivninger, der er ideelle til migrationsundersøgelser af denne art. Renset rotte oligodendrocytter blev derefter tilsat på renset rotte DRG axoner og induceret til myelinat. Efter at have bekræftet dannelsen af kompakt myelin, blev hgcs endelig føjet til co-kulturen og deres interaktioner med DRG axoner og oligodendrocytter blev overvåget i realtid ved hjælp af tidsforskudt mikroskopi. Under disse betingelser, hgcs danner tumor-lignende aggregat strukturer, der udtrykker GFAP og Ki67, migrere langs både myelinerede og ikke-myelerede axonal spor og interagere med disse axoner gennem dannelsen af pseudopodia. Vores ex vivo Co-Culture system kan bruges til at identificere nye cellulære og molekylære mekanismer af hGC migration og kunne potentielt anvendes til in vitro Drug effektivitet test.

For at studere samspillet mellem hgcs og axoner genererede vi rensede DRG axoner som tidligere beskrevet15,16,17,18. Disse rensede DRG axoner blev derefter seedet med hGCs, som dannede GFAP +/Ki67 + tumor lignende strukturer integreret i axonal Network, mens individuelle Hgc'er migrerede enten i forening eller mellem axonerne (figur 2). For at afgøre, hvordan hgcs interagerer ...

Watch this Scientific Journal Video about Real-Time Monitoring of Human Glioma Cell Migration on Dorsal Root Ganglion Axon-Oligodendrocyte Co-Cultures at JoVE.com

Real-Time Monitoring of Human Glioma Cell Migration on Dorsal Root Ganglion Axon-Oligodendrocyte Co-Cultures

Her præsenterer vi et ex-vivo blandet enkeltlags kultur system til studiet af human gliom Cell (HGC) migration i realtid. Denne model giver mulighed for at observere interaktioner mellem hGCs og både myelinerede og ikke-myelinerede axoner i et opdelt kammer.

Realtidsovervågning af menneskelige Glioma celle migration på dorsale root ganglion Axon-Oligodendrocyte Co-kulturer

Glioblastoma is one of the most aggressive human cancers due to extensive cellular heterogeneity and the migration properties of hGCs. In order to better ...

Denne ex vivo co-kultur system kan bruges til at identificere nye cellulære og molekylære mekanismer af human gliom celle migration og potentielt kunne anvendes til in vitro drug effekt test. Den største fordel ved denne teknik er evnen til at studere interaktioner mellem humane glioblastomceller og axoner i realtid. Demonstration af proceduren vil være John Zepecki, en kandidat studerende fra mit laboratorium.For at samle opdelte kulturretter fortyndes kollagenstrømsopløsningen til 500 mikrogram pr. milliliter i sterilt destilleret vand og blandes grundigt. Med en steril overførselpipette fyldes en 35 millimeter kulturskål med to milliliter af den fortyndede kollagenopløsning. Derefter lidt vippe skålen og fjerne opløsningen efterlader en tynd film af kollagen bag.Dispenser opløsningen i den næste 35 millimeter skål. Gentag denne proces, tilføje mere kollagen løsning efter behov, indtil alle retter er blevet belagt. For at polymerisere kollagen, i en laminar flow hætte, våde gaze puder med tre milliliter koncentreret ammoniumhydroxid og dække bakkerne i 15 minutter.Fjern gazepuder og lad de 35 millimeter retter tørre. Dernæst skal du bruge en metalfil til at arkivere punktet på en 18 gauge nål for at lave en stump spids. Læg nålen i blød i 70% ethanol til at sterilisere.Påfør silikonefedt på bunden af det ruminddemerede kammer. Sørg for, at fedtet er placeret pænt og overlapper i alle hjørner. Denne teknik kan være meget udfordrende, så vi foreslår, at du tager ekstra tid til at øve at anvende den korrekte mængde fedt, før du sætter de opdelte kamre for at sikre fremragende vækst.Det er også meget vigtigt at arbejde langsomt. Oprette flere kamre, end du tror, du får brug for, så du har ekstramateriale, hvis noget går galt. Tag låget af en 35 millimeter skål, inverter skålen og læg ridserne over kammeret.Tryk forsigtigt ned på bunden af skålen med et par vicesser. Når fedtkamrene påføres skålen, må du ikke trykke for hårdt. Ellers vil axonerne ikke være i stand til at vokse nedenunder i det tilstødende kammer.Brug de hæmotiske kraftas til forsigtigt at vende skålen over. Slip fortrivet. Placer en bunke fedt i bunden af midterrummet.Fyld hvert kammer med suppleret neuronal basal medium. Kontroller for utætheder og tætningslækt med silikonefedt efter behov. Fortsæt med at samle alle kulturretter og opbevar natten over ved 37 grader Celsius og 5% kuldioxid.Det opdelte kammer tillader brug af forskellige farmakologiske reagenser samt evnen til at sammenligne og kontrast resultater mellem to brønde på samme plade. For at frøe de forberedte kulturer, der indeholder en GBM neurosphere, skal mediet først udskiftes med suppleret NBF indeholdende 10% FBS i hvert distalt rum. Dernæst, med en P20 pipette indstillet til 10 mikroliter, tegne en GBM neurosphere fra kulturskålen.Placer spidsen af pipetten i det distale kammer tættest på midterkammeret uden at røre ved axonen og udvise GBM neurosphere langsomt, så den forsigtigt falder på axonerne. Lad kulturen blive i biosikkerhedsskabet i en time ved stuetemperatur, så GBM-neurosfæren kan fastgøres. Denne metode kan bruges til at teste farmakologiske forbindelser til behandling af hjernetumorer.Denne metode kan også anvendes på andre sygdomme i det perifere og centrale nervesystem såsom demyeliniserende neuropatier og multipel sklerose. I denne protokol, renset DRG axoner var seedet med HCGs, som dannede dobbelt positiv GFAP og KI67 tumor-lignende strukturer integreret i axonal netværk angivet af den røde tumor markører GFAP mens de enkelte HGCs udtrykke den grønne fluorescerende protein migreret enten i forening eller mellem axoner. Tilsætning af HFC'er på myelinerede DRG oligodendrocyte co-kulturer viste, at HGCs migrere i forbindelse med den røde farvede myelinerede axoner og væk fra tumormassen gennem dannelsen af pseudopodia.Når denne teknik blev perfektioneret, vi var i stand til at udnytte det til at studere migration af menneskelige GBM stamceller i realtid. Vi var også i stand til at studere effekter på cellemigration efter tilsætning af nye terapeutiske små molekylehæmmere. GBM kugle invaderer en myelinated oligodendrocyte DRG axon kultur er vist her.Den protokol, der er beskrevet her, kan danne grundlag for andre biomimetiske tredimensionelle ex vivo-systemer, der er designet til at vurdere virkningerne af nye lægemiddelbehandlinger. Man bør altid være forsigtig, når håndtering af humant afledte celler. Passende PV bør bæres, og blodbårne patogen uddannelse bør være afsluttet, så man forstår risikoen ved at arbejde med patient-afledte væv.Ammoniumhydroxid skal håndteres med forsigtighed, og du bør bære handsker, en laboratoriekittel, og arbejde i en hætte for at forhindre eksponering for dette kemikalie.

Research

JoVE Journal

Cancer Research

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma (DIPG)

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma DIPG

En protokol til Rapid Post-mortem Cell Culture af Diffus Intrinsic Pontine gliom (DIPG)

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a ...

Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts

Forberedelse og metaboliske analyse af 3-dimensionelle klumpformet Co kulturer af kræft i bugspytkirtlen celler og fibroblaster

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated ...

Using the Dot Assay to Analyze Migration of Cell Sheets

Ved hjælp af Dot analyse til at analysere Migration af celle ark

Although complex organisms appear static, their tissues are under a continuous turnover. As cells age, die, and are replaced by new cells, cells move ...

Serum and Plasma Copy Number Detection Using Real-time PCR

Serum og Plasma kopi nummer påvisning ved hjælp af Real-time PCR

Serum and plasma cell free DNA (cfDNA) has been shown as an informative, non-invasive source of biomarkers for cancer diagnosis, prognosis, monitoring, ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Realtid påvisning af i Vitro Tumor celle apoptose induceret af CD8+ T-celler til at studere immun suppressiv funktioner af Tumor infiltrerer myeloide celler

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures

Evaluering af biomarkører i gliom af Immunhistokemi på Paraffin-Embedded 3D gliom Neurosphere kulturer

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as ...

Time-Lapse 2D Imaging of Phagocytic Activity in M1 Macrophage-4T1 Mouse Mammary Carcinoma Cells in Co-cultures

Time-lapse 2D billeddannelse af Fagocytisk aktivitet i M1 Macrophage-4T1 mus mammary karcinom celler i co-kulturer

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer ...

Impedance-based Real-time Measurement of Cancer Cell Migration and Invasion

Impedans-baseret Real-time Måling af Kræft Cell Migration og Invasion

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These ...

Measuring Real-time Drug Response in Organotypic Tumor Tissue Slices

Måling Real-time Drug Response i organotypic tumorvæv skiver

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu ...

A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells

En hurtig screeningsarbejdsgang til identifikation af potentiel kombinationsbehandling for GBM ved hjælp af patientafledte gliomstamceller

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in ...