Name: hyaluronic-acid based hydrogels for 3-dimensional culture of patient-derived glioblastoma cells
Uploaded: 2018-08-24T14:30:00
Description: Watch this Scientific Journal Video about Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells at JoVE.com

Dette arbejde blev støttet med midler fra NIH (R21NS093199) og UCLA ARC 3R Award. Vores hjerteligste tak går til laboratoriet af Dr. Harley Kornblum til bestemmelse af HK301 og HK157 cellelinjer. Vi takker også UCLA væv patologi Core Laboratory (TPCL) for cryosectioning, avanceret lys mikroskopi/spektroskopi core facilitet (ALMS) i Californien nanosystemer Institute (CNSI) på UCLA for brug af en Konfokal mikroskop, UCLA Crump Institut for Molekylær billeddannelse til at bruge IVIS billedbehandlingssystem, UCLA molekylære Instrumentation Center (MIC) for at give Kernemagnetisk resonans-spektroskopi, og Flow flowcytometri kerne i Jonsson omfattende Cancer Center (JCCC) på UCLA for at levere instrumenterne til flow flowcytometri.

Alle menneskelige væv samling trin blev gennemført under institutionelt godkendte protokoller.
1. Thiolation af hyaluronsyre
Bemærk: Molære forhold er angivet med hensyn til antallet af carboxylat grupper, medmindre andet er angivet.
<ol><li>Opløse 500 mg natrium hyaluronate (HA, 500-750 kDa) på 10 mg/mL deioniseret vand, destilleret vand (DiH2O) i autoklave steriliseret, 250 mL Erlenmeyer-kolbe. Rør løsningen (~ 200 rpm) ved stuetemperatur i 2 ...

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogels+as+extracellular+matrix+mimics+for+3D+cell+culture.">Hydrogels as extracellular matrix mimics for 3D cell culture.</a> Biotechnology and bioengineering. 103 (4), 655-663 (2009).</li><li>Xiao, 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=Brain-mimetic+3d+culture+platforms+allow+investigation+of+cooperative+effects+of+extracellular+matrix+features+on+therapeutic+resistance+in+glioblastoma.">Brain-mimetic 3d culture platforms allow investigation of cooperative effects of extracellular matrix features on therapeutic resistance in glioblastoma.</a> Cancer Research. 78 (5), 1358-1370 (2018).</li><li>Holland, E. 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B., Kwak, H. J., Lee, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+hyaluronan+in+glioma+invasion.">Role of hyaluronan in glioma invasion.</a> Cell adhesion &amp; migration. 2 (3), 202-207 (2008).</li><li>Pedron, 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=Spatially+graded+hydrogels+for+preclinical+testing+of+glioblastoma+anticancer+therapeutics.">Spatially graded hydrogels for preclinical testing of glioblastoma anticancer therapeutics.</a> MRS communications. 7 (3), 442-449 (2017).</li><li>Jiglaire, C. J., 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=Ex+vivo+cultures+of+glioblastoma+in+three-dimensional+hydrogel+maintain+the+original+tumor+growth+behavior+and+are+suitable+for+preclinical+drug+and+radiation+sensitivity+screening.">Ex vivo cultures of glioblastoma in three-dimensional hydrogel maintain the original tumor growth behavior and are suitable for preclinical drug and radiation sensitivity screening.</a> Experimental cell research. 321 (2), 99-108 (2014).</li><li>Florczyk, S. J., 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=Porous+chitosan-hyaluronic+acid+scaffolds+as+a+mimic+of+glioblastoma+microenvironment+ECM.">Porous chitosan-hyaluronic acid scaffolds as a mimic of glioblastoma microenvironment ECM.</a> Biomaterials. 34 (38), 10143-10150 (2013).</li><li>Day, A. J., Prestwich, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronan-binding+proteins:+tying+up+the+giant.">Hyaluronan-binding proteins: tying up the giant.</a> Journal of Biological Chemistry. 277 (7), 4585-4588 (2002).</li><li>Wiranowska, M., Tresser, N., Saporta, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+interferon+and+anti-CD44+antibody+on+mouse+glioma+invasiveness+in+vitro.">The effect of interferon and anti-CD44 antibody on mouse glioma invasiveness in vitro.</a> Anticancer Research. 18 (5A), 3331-3338 (1998).</li><li>Charles, N. A., Holland, E. C., Gilbertson, R., Glass, R., Kettenmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+brain+tumor+microenvironment.">The brain tumor microenvironment.</a> GLIA. 59 (8), 1169-1180 (2011).</li><li>Gilg, A. G., 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=Targeting+hyaluronan+interactions+in+malignant+gliomas+and+their+drug-resistant+multipotent+progenitors.">Targeting hyaluronan interactions in malignant gliomas and their drug-resistant multipotent progenitors.</a> Clinical Cancer Research. 14 (6), 1804-1813 (2008).</li><li>Misra, S., Hascall, V. C., Markwald, R. R., Ghatak, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+hyaluronan+and+its+receptors+(CD44,+RHAMM)+regulate+the+activities+of+inflammation+and+cancer.">Interactions between hyaluronan and its receptors (CD44, RHAMM) regulate the activities of inflammation and cancer.</a> Frontiers in immunology. 6, 201 (2015).</li><li>Varga, I., 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=Expression+of+invasion-related+extracellular+matrix+molecules+in+human+glioblastoma+versus+intracerebral+lung+adenocarcinoma+metastasis.">Expression of invasion-related extracellular matrix molecules in human glioblastoma versus intracerebral lung adenocarcinoma metastasis.</a> Zentralblatt fur Neurochirurgie. 71 (4), 173-180 (2010).</li><li>Guo, W., Giancotti, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+signalling+during+tumour+progression.">Integrin signalling during tumour progression.</a> Nature Reviews Molecular Cell Biology. 5 (10), 816-826 (2004).</li><li>Bello, L., 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=&#945;v&#946;3+and+&#945;v&#946;5+integrin+expression+in+glioma+periphery.">&#945;v&#946;3 and &#945;v&#946;5 integrin expression in glioma periphery.</a> Neurosurgery. 49 (2), 380-390 (2001).</li><li>Chamberlain, M. C., Cloughsey, T., Reardon, D. A., Wen, P. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+treatment+for+glioblastoma:+integrin+inhibition.">A novel treatment for glioblastoma: integrin inhibition.</a> Expert review of neurotherapeutics. 12 (4), 421-435 (2012).</li><li>Chopra, 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=Augmentation+of+integrin-mediated+mechanotransduction+by+hyaluronic+acid.">Augmentation of integrin-mediated mechanotransduction by hyaluronic acid.</a> Biomaterials. 35 (1), 71-82 (2014).</li><li>Kim, Y., Kumar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD44-mediated+adhesion+to+hyaluronic+acid+contributes+to+mechanosensing+and+invasive+motility.">CD44-mediated adhesion to hyaluronic acid contributes to mechanosensing and invasive motility.</a> Molecular Cancer Research. 12 (10), 1416-1429 (2014).</li><li>Joo, K. M., 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=Patient-specific+orthotopic+glioblastoma+xenograft+models+recapitulate+the+histopathology+and+biology+of+human+glioblastomas+in+situ.">Patient-specific orthotopic glioblastoma xenograft models recapitulate the histopathology and biology of human glioblastomas in situ.</a> Cell Reports. 3 (1), 260-273 (2013).</li><li>Oh, Y. T., 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=Translational+validation+of+personalized+treatment+strategy+based+on+genetic+characteristics+of+glioblastoma.">Translational validation of personalized treatment strategy based on genetic characteristics of glioblastoma.</a> PloS one. 9 (8), e103327 (2014).</li><li>Pedron, S., Becka, E., Harley, B. A. C. <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+glioma+cell+phenotype+in+3D+matrices+by+hyaluronic+acid.">Regulation of glioma cell phenotype in 3D matrices by hyaluronic acid.</a> Biomaterials. 34 (30), 7408-7417 (2013).</li><li>Wang, C., Tong, X., Yang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioengineered+3D+brain+tumor+model+to+elucidate+the+effects+of+matrix+stiffness+on+glioblastoma+cell+behavior+using+PEG-based+hydrogels.">Bioengineered 3D brain tumor model to elucidate the effects of matrix stiffness on glioblastoma cell behavior using PEG-based hydrogels.</a> Molecular pharmaceutics. 11 (7), 2115-2125 (2014).</li><li>Zhu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioactive+modification+of+poly(ethylene+glycol)+hydrogels+for+tissue+engineering.">Bioactive modification of poly(ethylene glycol) hydrogels for tissue engineering.</a> Biomaterials. 31 (17), 4639-4656 (2010).</li><li>Stern, R., Asari, A. 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Generation af reproducerbare data ved hjælp af denne 3D kultur system kræver: 1) konsekvent batch til batch thiolation ha, 2) praksis til at opnå effektiv blanding af hydrogel prækursorer og håndtering af hydrogel kulturer for at forhindre skader og 3) optimeret såning tæthed for hver cellelinje bruges.
Når en bestemt vægt procentdel af HA ønskes i hydrogel, bestemmer graden af thiolation ha bitmapgenkendelse tæthed. Vi anbefaler at bruge en konsekvent mængde af HA for hver enkelt ...

Tre-dimensionelle (3D) kultur systemer sammenfatte interaktioner mellem cellerne og deres omgivende ekstracellulære matrix (ECM) i native væv bedre end deres todimensionale (2D) modparter1,2. Fremskridt i vævsmanipulering har givet avancerede, 3D kultur platforme, der aktiverer kontrollerede undersøgelser af 1) hvordan kemiske og fysiske komponenter af matrix mikromiljø påvirker celle adfærd og 2) virkning af nye terapeutiske strategier for en række sygdomme, herunder kræft2. Mens in vitro- modeller kan ikke højde for systemiske faktorer, såsom hormonforstyrrende og immun signaler, og dermed kan ikke helt erstatte i vivo modeller, giver de flere fordele, herunder reproducerbarhed, eksperimentelle kontrol, overkommelige priser og hastighed. Her, beskriver vi brugen af hjerne-mimetiske hydrogels i hvilke 3D kulturer af patient-afledte hjerne tumorceller fange mange aspekter af tumor fysiologi, navnlig dynamikken i erhverve behandling modstand3. I forhold til andre in vitro- metoder, repræsenterer disse kulturer bedre i vivo tumor modeller og kliniske observationer3.
Glioblastom (GBM) er den mest hyppige og dødelig kræft med oprindelse i hjernen, med en median overlevelse på kun 1-2 år4,5. I de seneste år, har mange undersøgelser fokuseret på indflydelse af tumor matrix miljø i GBM6,7,8. Unikke hjernen ECM er blevet rapporteret at påvirke GBM celle migration, spredning og terapeutiske modstand6,7,8,9,10,11 , 12. Hyaluronic syre (HA) er en rigelige glykosaminoglykan (GAG) i hjernen, hvor det interagerer med andre GAGs og proteoglycaner at danne en hydrogel-lignende mesh13. Mange undersøgelser har rapporteret HA overekspression i GBM tumorer og dens efterfølgende virkninger på kræft progression8,9,13,14,15,16 ,17. Andre ECM komponenter også påvirke GBM tumor vækst og invasion6,7,15,18. For eksempel, fibronektin, og vitronectin, som er typisk overexpressed i GBM, fremkalde heterodimerization celle langlinefartøjer integrin receptorer gennem binding til "M.H.T." sekvens og indlede komplekse signaling cascades, der fremmer tumor overlevelse19 ,20,21. Udover biokemiske påvirkninger påvirke fysiske egenskaber af matrixen væv også GBM progression22,23.
Løbende erhvervelse af modstand på terapier er en af de vigtigste drivkræfter bag GBM dødelighed4. Narkotika viser lovende resultater i 2D eller gliomasphere modeller har undladt i efterfølgende dyreforsøg og kliniske tilfælde3, som angiver, at virkningerne af microenvironmental faktorer bidraget betydeligt til GBM tumor svar1. Mens dyremodeller kan give en 3D, fysiologisk fald mikromiljø til xenografted patient celler og generere klinisk relevante resultater24,25, kompleksiteten af hjernen mikromiljø i vivo gør det udfordrende for at afgøre, hvilke funktioner, herunder celle-matrix interaktioner, er nøglen til specifikke biologiske resultater. Identifikation af nye terapeutiske mål vil drage fordel af brugen af forenklede kultur platforme som biokemiske og biofysiske egenskaber er defineret.
I modsætning til tidligere rapporteret biomateriale modeller af GBM tumor mikromiljø26,27 , som ikke har opnået ægte ortogonale kontrol over individuelle biokemiske og fysiske funktioner af ECM, biomateriale platform rapporteret her giver afkoblingen af bidragene fra flere uafhængige funktioner til GBM celle fænotype. Her præsenterer vi en HA-baseret, retvinklet afstemmelige, hydrogel system for 3D kultur af patient-afledte GBM celler. Hydrogels er dannet af to polymer komponenter: 1) biologisk aktive HA og 2) biologisk inaktivt polyethylenglycol (PEG). PIND er en udbredt biokompatible og hydrofile materiale med lav protein adsorption og minimal immunogenicitet28. Her, er ca. 5% af glucuronsyre syre fraspaltning på HA kæder functionalized med thiol grupper at aktivere crosslinking til et kommercielt tilgængelige 4-arm-PIND blev afsluttet med maleimides via Michael-type tilsætning. I sin mest almindelige form i kroppen findes HA i høj molekylvægt (HMW) kæder. Her, en lav grad af modifikation af HMW HA (500-750 kDa) hjælper med at bevare indfødte vekselvirkninger mellem HA og dets cellereceptorer, herunder CD4429. Ved at erstatte PIND-thiol for HA-thiol samtidig opretholde et konstant molære forhold på samlede dithioler til maleimides, kan HA koncentration være afkoblet fra mekaniske egenskaber af de resulterende hydrogels. Støkiometriske kontrolelementer kan desuden bruges til at konjugat cystein-afsluttet peptider til en defineret gennemsnitlige antal maleimide-afsluttet arme på hver 4-arm-PIND. Indarbejdelse af ECM-afledte, selvklæbende peptider muliggør interaktion med integriner på kulturperler celler, hvorigennem biokemiske og kemiske signaler er transduced1. Maleimide-thiol tilføjelse forekommer meget hurtigt under fysiologiske tilstande, minimere den tid, der kræves for celle indkapsling og maksimere overlevelse af patient-afledte celler. Derudover hydrogel kulturer kan blive behandlet som typisk væv modellen og er kompatibel med standard karakterisering teknikker herunder Western blotting, flowcytometri og immunfluorescens farvning. Følgende protokol beskriver procedurerne for opdigte hydrogels, oprettelse af 3D kulturer af patient-afledte GBM celler og teknikker til biokemiske analyser.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">pH meter</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">N/A</td>
			<td style="width:1579px;">Any pH meter that has pH 2-10 sensitivity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Stir plate</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">N/A</td>
			<td>General lab equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Erlenmeyer flask (125mL)</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">FB-501-125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">dialysis tubes</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">21-152-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">2L polypropylene beaker</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">S01916</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">sodium hyaluronan</td>
			<td style="width:136px;">Lifecore</td>
			<td style="width:121px;">HA700k-5</td>
			<td>500-750 kDa range</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">PI-22980</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">N-hydroxysuccinimide (NHS)</td>
			<td style="width:136px;">sigma aldrich</td>
			<td style="width:121px;">130672-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Hydrochloric acid (HCl)</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">SA48-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Sodium hydroxide (NaOH)</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">SS266-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Cystamine dihydrochloride</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">AC111770250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Dithiolthreitol (DTT)</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">BP172-25</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:368px;">Ellman&#39;s test reagent (5-(3-Carboxy-4-nitrophenyl)disulfanyl-2-nitrobenzoic acid</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td style="width:121px;">D218200-1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Deuterated water (deuterium oxide)</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">AC166301000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">0.22&#181;m vacuum driven filter</td>
			<td style="width:136px;">CellTreat</td>
			<td style="width:121px;">229706</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Phosphate buffered saline (PBS)</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">P32080-100T</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Hanks&#39; balanced salt saline (HBSS)</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">MT-21-022-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">4-arm-PEG-maleimide</td>
			<td style="width:136px;">JenKem Technology</td>
			<td style="width:121px;">A7029-1</td>
			<td>molecular weight around 20kDa</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">4-arm-PEG-thiol</td>
			<td style="width:136px;">JenKem Technology</td>
			<td style="width:121px;">A7039-1</td>
			<td>molecular weight around 20kDa</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">L-Cysteine&#160;</td>
			<td style="width:136px;">sigma aldrich</td>
			<td style="width:121px;">C7880-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">RGD ECM mimetic peptide</td>
			<td style="width:136px;">Genscript Biotech</td>
			<td style="width:121px;">N/A</td>
			<td>Custom peptide with sequence &#34;GCGYGRGDSPG&#34;, N-terminal should be acetylated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">silicone molds</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td style="width:121px;">GBL664201-25EA</td>
			<td>Use razor blade to cut into single pieces</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">complete culture medium</td>
			<td style="width:136px;">Various</td>
			<td style="width:121px;">Various</td>
			<td>DMEM/F12 (Thermofisher) with non-serum supplement (G21 from GeminiBio), epidermal growth factor 50ng/mL (Peprotech), fibroblast growth factor 20ng/mL (Pepro Tech) and heprain 25&#181;g/mL (Sigma Aldrich), culture medium varies in different labs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">patient derived GBM cell</td>
			<td style="width:136px;">N/A</td>
			<td style="width:121px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">20G needle</td>
			<td style="width:136px;">BD medical</td>
			<td style="width:121px;">305175</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">1mL syringe</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">14-823-434</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">10mL syringe</td>
			<td style="width:136px;">BD medical</td>
			<td style="width:121px;">302995</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">RIPA Buffer</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">PI-89901</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">protease/phosphatase inhibitor mini tablet</td>
			<td style="width:136px;">sigma aldrich</td>
			<td style="width:121px;">5892970001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">vortex shaker</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">12-814-5Q</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">TrypLE express</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">12604013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">70&#181;m cell strainer</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">22-363-548</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Paraformaldehyde</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">AC416785000</td>
			<td>Dissolve 4% (w/v) in PBS, keep pH 7.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">D-sucrose</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">BP220-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Optimal Cutting Temperature (O.C.T.) compound</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">NC9373881</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Cell culture incubator</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">N/A</td>
			<td>Any General One with 5% CO2 and 37C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">fridge/freezer</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">N/A</td>
			<td>Any General Lab equipment with -20C and -80C capacity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Disposable embedding molds</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">12-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Lyapholizer</td>
			<td style="width:136px;">Labconco</td>
			<td style="width:121px;">N/A</td>
			<td>Any -105C freeze dryers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">HEPES</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">BP310-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Amber vial</td>
			<td style="width:136px;">Kimble Chase</td>
			<td style="width:121px;">60912D-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Wide orifice pipette tips</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">9405120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">2-methylbutane</td>
			<td style="width:136px;">Thermo Fisher</td>
			<td style="width:121px;">03551-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Dry Ice</td>
			<td style="width:136px;">N/A</td>
			<td style="width:121px;">N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

hyaluronic-acid based hydrogels for 3-dimensional culture of patient-derived glioblastoma cells

Glioblastom (GBM) er den mest almindelige, men mest dødbringende, centralnervesystemet kræft. I de seneste år, har mange undersøgelser fokuseret på hvordan den ekstracellulære matrix (ECM) af unikke hjernen miljø, såsom hyaluronsyre (HA), letter GBM progression og invasion. Men de fleste in vitro- kultur modeller omfatter GBM celler uden for rammerne af en ECM. Murine xenografts GBM celler anvendes almindeligt som godt. Men, i vivo modeller gør det vanskeligt at isolere bidrag fra individuelle funktioner af komplekse tumor mikromiljø til tumor adfærd. Her, beskriver vi en HA hydrogel-baseret, tre-dimensionelle (3D) kultur platform, der giver forskere til selvstændigt alter HA koncentration og stivhed. Høj molekylvægt HA og polyethylenglycol (PEG) omfatter hydrogels, som er crosslinked via Michael-type tilsætning i overværelse af levende celler. 3D hydrogel kulturer af patient-afledte GBM celler udstille levedygtighed og spredning satser så godt som eller bedre end, når kulturperler som standard gliomaspheres. Hydrogel system sætter også inkorporering af ECM-mimetiske peptider til at isolere effekten af specifik celle-ECM interaktioner. Hydrogels er optisk gennemsigtig, så levende celler kan være afbildet i 3D kultur. Endelig er HA hydrogel kulturer kompatibel med standardteknikker for molekylære og cellulære analyser, herunder PCR, Western blotting og cryosectioning efterfulgt af immunofluorescens farvning.

For hvert parti af thiolated HA, bør graden af thiolation kontrolleres ved hjælp af H1-NMR eller en Ellman test. HA ændring ved hjælp af proceduren beskrevet her konsekvent genererer ~ 5% thiolation (defineret som den molære forhold af dithioler at HA disaccharider) (figur 1).
Opsætning af denne nye kultur platform vil kræve hvert laboratorium til at udføre streng...

Watch this Scientific Journal Video about Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells at JoVE.com

Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells

Vi præsenterer her, en protokol for tre-dimensionelle kultur af patient-afledte glioblastom celler i retvinklet afstemmelige biomaterialer designet til at efterligne den hjerne matrix. Denne fremgangsmåde giver en in vitro-, eksperimentelle platform, der vedligeholder mange egenskaber i vivo glioblastom celler typisk tabt i kulturen.

Hyaluronsyre baseret Hydrogels til 3-dimensionelle kultur af Patient-afledte glioblastom celler

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular ...

Research

JoVE Journal

Bioengineering

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

Roterende Cell Culture Systems for Human Cell Culture: Human trofoblasten celler som en model

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

Dyrkning af humane neurale stamceller i en 3-dimensionel selvsamlende peptid Hydrogel

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Selvrapportering Stilladser for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

Syntesen af ​​RGD-funktionaliserede Hydrogeler som værktøj for terapeutiske applikationer

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Light-medieret dannelse og mønsterdannelse af hydrogeler til celledyrkningsapplikationer

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...