Name: generation of microtumors using 3d human biogel culture system and patient-derived glioblastoma cells for kinomic profiling and drug response testing
Uploaded: 2016-06-09T15:00:00
Description: Watch this Scientific Journal Video about Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing at JoVE.

Soutenu par subvention du NIH R21 (PI: C. Willey, CA185712-01), Brain Tumor sentence SPORE (PD: GY Gillespie, P20CA 151129-03) et le contrat SBIR (PI: R. Singh, N43CO-2013-00026).

NOTE: Toutes les évaluations de thérapie xénogreffes ont été effectuées en utilisant un modèle de tumeur orthotopique pour glioblastome sur un protocole approuvé par le Comité soin et l&#39;utilisation des animaux institutionnels. 1. Isolement des cellules GBM xénogreffes patients dérivés <ol><li> Préparation des réactifs <ol><li> Reconstituer collagénase I dans de l&#39;eau stérile à une concentration de 5 mg / ml et stérile filtre. Magasin à aliquotes ...

<ol>
	<li>Ohgaki, H., Kleihues, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Population-based+studies+on+incidence,+survival+rates,+and+genetic+alterations+in+astrocytic+and+oligodendroglial+gliomas.">Population-based studies on incidence, survival rates, and genetic alterations in astrocytic and oligodendroglial gliomas.</a> J Neuropathol Exp Neurol. 64 (6), 479-489 (2005).</li><li>Wen, P. Y., Kesari, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malignant+gliomas+in+adults.">Malignant gliomas in adults.</a> N Engl J Med. 359 (5), 492-507 (2008).</li><li>Thumma, S. 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=Effect+of+pretreatment+clinical+factors+on+overall+survival+in+glioblastoma+multiforme:+a+Surveillance+Epidemiology+and+End+Results+(SEER)+population+analysis.">Effect of pretreatment clinical factors on overall survival in glioblastoma multiforme: a Surveillance Epidemiology and End Results (SEER) population analysis.</a> World J Surg Oncol. 10 (75), (2012).</li><li>Furnari, F. B., Cloughesy, T. F., Cavenee, W. K., Mischel, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+epidermal+growth+factor+receptor+signalling+networks+in+glioblastoma.">Heterogeneity of epidermal growth factor receptor signalling networks in glioblastoma.</a> Nat Rev Cancer. 15 (5), 302-310 (2015).</li><li>Mischel, P. S., Cloughesy, T. F., Nelson, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-microarray+analysis+of+brain+cancer:+molecular+classification+for+therapy.">DNA-microarray analysis of brain cancer: molecular classification for therapy.</a> Nat Rev Neurosci. 5 (10), 782-792 (2004).</li><li>Verhaak, R. 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=Integrated+genomic+analysis+identifies+clinically+relevant+subtypes+of+glioblastoma+characterized+by+abnormalities+in+PDGFRA,+IDH1,+EGFR,+and+NF1.">Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1.</a> Cancer Cell. 17 (1), 98-110 (2010).</li><li>De Witt Hamer, P. 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=The+genomic+profile+of+human+malignant+glioma+is+altered+early+in+primary+cell+culture+and+preserved+in+spheroids.">The genomic profile of human malignant glioma is altered early in primary cell culture and preserved in spheroids.</a> Oncogene. 27 (14), 2091-2096 (2008).</li><li>Shankavaram, U. 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=Molecular+profiling+indicates+orthotopic+xenograft+of+glioma+cell+lines+simulate+a+subclass+of+human+glioblastoma.">Molecular profiling indicates orthotopic xenograft of glioma cell lines simulate a subclass of human glioblastoma.</a> J Cell Mol Med. 16 (3), 545-554 (2012).</li><li>Abbott, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture:+biology's+new+dimension.">Cell culture: biology's new dimension.</a> Nature. 424 (6951), 870-872 (2003).</li><li>Rao, S. 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 Eng Part B Rev. 20 (4), 314-327 (2014).</li><li>Pampaloni, F., Reynaud, E. G., Stelzer, E. 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+third+dimension+bridges+the+gap+between+cell+culture+and+live+tissue.">The third dimension bridges the gap between cell culture and live tissue.</a> Nat Rev Mol Cell Biol. 8 (10), 839-845 (2007).</li><li>Hollister, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porous+scaffold+design+for+tissue+engineering.">Porous scaffold design for tissue engineering.</a> Nat Mater. 4 (7), 518-524 (2005).</li><li>Rowley, J. A., Madlambayan, G., Mooney, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alginate+hydrogels+as+synthetic+extracellular+matrix+materials.">Alginate hydrogels as synthetic extracellular matrix materials.</a> Biomaterials. 20 (1), 45-53 (1999).</li><li>Hughes, C. S., Postovit, L. M., Lajoie, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrigel:+a+complex+protein+mixture+required+for+optimal+growth+of+cell+culture.">Matrigel: a complex protein mixture required for optimal growth of cell culture.</a> Proteomics. 10 (9), 1886-1890 (2010).</li><li>Yoshino, J. E., 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=Proliferation+and+differentiation+of+a+transfected+Schwann+cell+line+is+altered+by+an+artificial+basement+membrane.">Proliferation and differentiation of a transfected Schwann cell line is altered by an artificial basement membrane.</a> Glia. 3 (5), 315-321 (1990).</li><li>Siegal, G. P., Singh, R., Foundation, T. U. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biologically+active+native+biomatrix+composition.">Biologically active native biomatrix composition.</a> US patent. , (2010).</li><li>Sarkaria, J. N., 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=Use+of+an+orthotopic+xenograft+model+for+assessing+the+effect+of+epidermal+growth+factor+receptor+amplification+on+glioblastoma+radiation+response.">Use of an orthotopic xenograft model for assessing the effect of epidermal growth factor receptor amplification on glioblastoma radiation response.</a> Clin Cancer Res. 12 (7), 2264-2271 (2006).</li><li>Strober, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypan+Blue+Exclusion+Test+of+Cell+Viability.">Trypan Blue Exclusion Test of Cell Viability.</a> Curr Protoc Immunol. 111, 1-3 (2015).</li><li>Anderson, J. 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=Kinomic+exploration+of+temozolomide+and+radiation+resistance+in+Glioblastoma+multiforme+xenolines.">Kinomic exploration of temozolomide and radiation resistance in Glioblastoma multiforme xenolines.</a> Radiother Oncol. 111 (3), 468-474 (2014).</li><li>Anderson, J. 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=Kinomic+profiling+of+electromagnetic+navigational+bronchoscopy+specimens:+a+new+approach+for+personalized+medicine.">Kinomic profiling of electromagnetic navigational bronchoscopy specimens: a new approach for personalized medicine.</a> PLoS One. 9 (12), 116388 (2014).</li><li>Jarboe, J. 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=Kinomic+profiling+approach+identifies+Trk+as+a+novel+radiation+modulator.">Kinomic profiling approach identifies Trk as a novel radiation modulator.</a> Radiother Oncol. 103 (3), 380-387 (2012).</li><li>Anderson, J. 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=High+Throughput+Kinomic+Profiling+of+Human+Clear+Cell+Renal+Cell+Carcinoma+Identifies+Kinase+Activity+Dependent+Molecular+Subtypes.">High Throughput Kinomic Profiling of Human Clear Cell Renal Cell Carcinoma Identifies Kinase Activity Dependent Molecular Subtypes.</a> PLoS One. 10 (9), 0139267 (2015).</li><li>Anderson, J. 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=Kinomic+Alterations+in+Atypical+Meningioma.">Kinomic Alterations in Atypical Meningioma.</a> Medical Research Archives. 3, (2015).</li><li>Hothi, P., 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=High-throughput+chemical+screens+identify+disulfiram+as+an+inhibitor+of+human+glioblastoma+stem+cells.">High-throughput chemical screens identify disulfiram as an inhibitor of human glioblastoma stem cells.</a> Oncotarget. 3 (10), 1124-1136 (2012).</li><li>Quartararo, C. E., Reznik, E., deCarvalho, A. C., Mikkelsen, T., Stockwell, 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=High-Throughput+Screening+of+Patient-Derived+Cultures+Reveals+Potential+for+Precision+Medicine+in+Glioblastoma.">High-Throughput Screening of Patient-Derived Cultures Reveals Potential for Precision Medicine in Glioblastoma.</a> ACS Med Chem Lett. 6 (8), 948-952 (2015).</li><li>Ma, 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=Towards+personalized+medicine+with+a+three-dimensional+micro-scale+perfusion-based+two-chamber+tissue+model+system.">Towards personalized medicine with a three-dimensional micro-scale perfusion-based two-chamber tissue model system.</a> Biomaterials. 33 (17), 4353-4361 (2012).</li><li>Pedron, S., Becka, E., Harley, 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=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>Rape, A., Ananthanarayanan, B., 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=Engineering+strategies+to+mimic+the+glioblastoma+microenvironment.">Engineering strategies to mimic the glioblastoma microenvironment.</a> Adv Drug Deliv Rev. 79-90, 172-183 (2014).</li><li>Willey, C. D., Gilbert, A. N., Anderson, J. C., Gillespie, G. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-Derived+Xenografts+as+a+Model+System+for+Radiation+Research.">Patient-Derived Xenografts as a Model System for Radiation Research.</a> Semin Radiat Oncol. 25 (4), 273-280 (2015).</li></ol>

Les étapes critiques au sein du protocole portent essentiellement sur microtumor génération, ainsi que dosage des médicaments et de l&#39;entretien. Parce que les perles de microtumor sont fragiles et facilement déchiré, un soin extrême est nécessaire dans les deux stades de développement d&#39;un essai et d&#39;entretien. Si une erreur se produit au cours de l&#39;un de ces procédés, l&#39;interprétation expérimentale peut être compromise, ce qui provoque l&#39;extension ou la répétition inutile des exp...

Les tumeurs les plus courantes primaires intracrâniennes cérébrales malignes sont de grade III et astrocytomes de grade IV glioblastome (glioblastome ou GBM). Ces tumeurs offrent un mauvais pronostic avec la médiane de survie d&#39; un an entre 12 - 15 mois avec les thérapies actuelles pour GBM aux États - Unis 1-3. multimodales thérapies comprennent la chirurgie, la radiothérapie et la chimiothérapie, y compris le témozolomide (TMZ) et les agents de la kinase ciblée. la signalisation de la kinase est souvent dérégulé dans la GBM, y compris les sous-ensembles de tumeurs avec une amplification ou des mutations d&#39;activation de la croissance du récepteur du facteur épidermique (EGFR), l&#39;augmentation du Platelet Derived Growth Factor Receptor (PDGFR) de signalisation, l&#39;augmentation de phosphatidylinositol-3 kinase (PI3K), et tumeur supportant une signalisation à travers l&#39; endothélium vasculaire angiogénique Growth Factor Receptor (VEGFR), ainsi que d&#39; autres kinases entraîné voies 4-6. Current in vitro et in vivo dans des modèles perdent souvent ces altérations représentatives &lt;sup&gt; 7. En outre, le profilage génétique n&#39;a pas offert les avantages escomptés qui peuvent refléter le fait que les changements génétiques et épigénétiques ne prédisent pas toujours des changements au niveau de l&#39;activité de la protéine, où la plupart kinase agents de ciblage agissent directement, et où les thérapies avec d&#39;autres mécanismes d&#39;action peuvent agir indirectement. La ligne traditionnelle de cellules immortalisées qui peut être repiqué ad infinitum a longtemps été la norme pour le dépistage des drogues en raison de leur facilité d&#39;entretien et de reproductibilité. Toutefois, ce modèle souffre d&#39;un environnement de croissance élevé en éléments nutritifs (et artificielle) qui sélectionne les cellules qui diffèrent grandement de la tumeur d&#39;origine en croissance rapide. En tant que tel, il y a eu un intérêt considérable dans le développement de systèmes de modèles plus réalistes qui reflètent un système biologique de la tumeur plus complexe est présent chez le patient. xénogreffes de tumeurs développées directement à partir d&#39;une tumeur primaire développée chez la souris ( &quot;xenoline&quot; xénogreffe dérivé du patient ou PDX) disposide système modèle plus représentatif, en particulier dans le cadre de traitements contre le cancer, comme ils se font sentir de prédire de manière plus fiable succès clinique. 8 Malgré la biologie plus réfléchi, ces modèles sont coûteux et sont difficiles à établir et à maintenir. De plus, ils ne se prêtent pas à des études à haut débit. La nécessité de mieux développer des modèles biologiques qui reflètent plus précisément les altérations moléculaires dans les tumeurs primaires et de profil et tester ces modèles en utilisant des mesures directes de l&#39;activité kinase, pas marqueurs substituts génétiques, est clair. Il est bien reconnu que , contrairement à deux dimensions (2D) des cultures monocouches, 3D ou modèles d&#39;analyse multicellulaires peuvent fournir plus physiologiquement effets pertinents 9-11. culture 3D commune approches impliquent des microsupports de matrice revêtu et la formation de sphéroïde cellulaire. sphéroïdes de tumeur peuvent être générés via l&#39;agrégation cellulaire en utilisant ballon rotatif, plaque pHEMA et la pendaison des techniques de chute. Limitations de tapproches es comprennent: l&#39;incapacité de certaines cellules pour former des sphéroïdes stables, la variabilité de la croissance et les défis avec les types de cellules mixtes. Sinon, beaucoup synthétique (hydrogel, polymère) et d&#39; origine animale Engelbreth-Holm-Swarm (EHS) matrice à partir de sarcomes de souris, le collagène bovin) matrices ont été développés pour la culture 3D études 12-14. Matrice de souris EHS est largement utilisée mais connue pour favoriser la croissance et la différenciation cellulaires in vitro et in vivo 15. Afin de reproduire la biologie de la tumeur en 3D, un système de biomatrice humain a été développé par le Dr Raj Singh et al. 16. La libre facteur biogel humain naturel, la croissance permet échafauds de culture 3D (perles, disques), qui soutiennent la culture à long terme de plusieurs types de cellules. Une série de 3D biogel humaine conceptions de la culture sont mis en place pour étudier la croissance tumorale, l&#39;adhérence, l&#39;angiogenèse et l&#39;invasion des propriétés. Avantages et propriétés de biogel humaine par rapport à commungels de souris EHS sont résumés dans le tableau 1 et le tableau 2. <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> La source: </td><td> Amnios humain (tissus Pooled) libre Pathogen, IRB-exemptés / approuvés </td></tr><tr><td> Nature ECM: </td><td> Non dénaturé Biogel (GLP-production) </td></tr><tr><td> Clé Composants: </td><td> Col-I (38%), la laminine (22%), Col-IV (20%), Col-III (7%), entactine &amp; HSPG (&lt;3%) </td></tr><tr><td> Gratuit GF-: </td><td> Indétectable EGF, FGF, TGF, VEGF, PDGF (non-angiogénique, non toxique) </td></tr></tbody></table> Tableau 1: Propriétés de Biogel humain par rapport à EHS Gels communs. <table border="1" fo:keep-together.within-page="1" fo:keep-wie-next.within page = &quot;always&quot;&gt; <tbody><tr><td> Biogel humain </td><td> gels EHS </td></tr><tr><td> Matrice humaine naturelle </td><td> Matrice de la souris reconstituée </td></tr><tr><td> la croissance et la différenciation cellulaire contrôlée </td><td> Peut promouvoir la croissance cellulaire et la différenciation </td></tr><tr><td> l&#39;expression du gène Physiologic </td><td> l&#39;expression du gène variable </td></tr><tr><td> modèle de culture 3D tissus comme </td><td> modèle de culture à base Plate- </td></tr></tbody></table> Tableau 2: Avantages de Biogel humain par rapport à EHS Gels communs.

<table border="0" cellpadding="0" cellspacing="0" width="1142">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Collagenase-I&#160;</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">CO130</td>
			<td style="width:496px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Trypsin EDTA (10X)</td>
			<td style="width:184px;">Invitrogen</td>
			<td style="width:119px;">15400-054&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Neurobasal-A</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">10888-022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">N-2 Supplement</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">17502-048</td>
			<td>1x final concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">B-27 Supplement w/o Vitamin A</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">12587-010</td>
			<td>1x final concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Recombinant Human FGF-basic</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">PHG0266</td>
			<td>10 ng/mL final concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Recombinant Human EGF</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">PGH0315</td>
			<td>10 ng/mL final concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">L-Glutamine</td>
			<td style="width:184px;">Corning Cellgro Mediatech</td>
			<td style="width:119px;">25-005-CI</td>
			<td>2 mM final concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Fungizone</td>
			<td style="width:184px;">Omega Scientific</td>
			<td style="width:119px;">FG-70</td>
			<td>2.5 ug/mL final concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Penicillin Streptomycin</td>
			<td style="width:184px;">Omega Scientific</td>
			<td style="width:119px;">PS-20</td>
			<td>100 U/mL Penicillin G, 100 ug/mL Streptomycin final concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Gentamicin</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">15750-060</td>
			<td>50 ng/mL final concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">MTT</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">M6494</td>
			<td>prepared to 5 mg/mL in PBS and sterile filtered, 1 mg/mL in well</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">SDS</td>
			<td style="width:184px;">Fisher</td>
			<td style="width:119px;">BP166</td>
			<td>for MTT lysis buffer, prepared to 10% in 0.01M HCL, 5% in well</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">HCl</td>
			<td style="width:184px;">Fisher</td>
			<td style="width:119px;">A144SI-212</td>
			<td>for MTT lysis buffer, prepared to 0.01M with SDS, 5 mM in well</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Calcein AM</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">C1430</td>
			<td>1 mM in DMSO stock, 2 uM in PBS staining solution, 1 uM in well</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Halt&#8217;s Protein Phosphatase Inhibitor cocktail&#160;</td>
			<td style="width:184px;">Pierce ThermoScientific</td>
			<td style="width:119px;">78420</td>
			<td>1:100 ratio in MPER&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Halt&#39;s Protein Protease Inhibitor&#160;</td>
			<td style="width:184px;">Pierce ThermoScientific</td>
			<td style="width:119px;">87786</td>
			<td>1:100 ratio in MPER</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Mammalian Protein Extraction Reagent (MPER)</td>
			<td style="width:184px;">Pierce ThermoScientific</td>
			<td style="width:119px;">PI78501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Trypan Blue</td>
			<td style="width:184px;">Pierce ThermoScientific</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">DMSO</td>
			<td style="width:184px;">Fisher</td>
			<td style="width:119px;">BP231</td>
			<td>for dissolution of calcein AM &#38; compounds</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Phosphate-Buffered Saline without Ca/Mg</td>
			<td style="width:184px;">Lonza</td>
			<td style="width:119px;">17-517Q</td>
			<td>diluted to 1X with MiliQ ultrapure water and sterile filtered (for cell culture)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Dulbecco&#39;s Phosphate-Buffered Saline with Ca/Mg</td>
			<td style="width:184px;">Corning Cellgro Mediatech</td>
			<td style="width:119px;">20-030-CV</td>
			<td>diluted to 1X with MiliQ ultrapure water (for pre-fixation wash)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">10% Neutral Buffered Formalin</td>
			<td style="width:184px;">Protocol</td>
			<td style="width:119px;">032-060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Trypan Blue</td>
			<td style="width:184px;">Pierce ThermoScientific</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">High Density Hubiogel</td>
			<td style="width:184px;">Vivo Biosciences</td>
			<td style="width:119px;">HDHG-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Halt&#39;s Protein Phosphatase Inhibitor</td>
			<td style="width:184px;">Pierce</td>
			<td style="width:119px;">78420</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Halt&#39;s Protein Protease Inhibitor</td>
			<td style="width:184px;">Pierce</td>
			<td style="width:119px;">87786</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Mammalian Protein Extraction Reagent (MPER)</td>
			<td style="width:184px;">Thermo Scientific</td>
			<td style="width:119px;">78501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Protein Tyrosine Kinase (PTK) Array Profiling chip</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;">86312</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">PTK kinase buffer</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;">36000</td>
			<td>300 &#181;l 10X PK buffer stock in 2.7 ml dH20, catalog number for PTK reagent kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">ATP</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;">36000</td>
			<td>catalog number for PTK reagent kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PY20- FITC-conjugated antibody</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;">36000</td>
			<td>catalog number for PTK reagent kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PTK Additive</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;">32114</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PTK-MM1 tube (10X BSA)</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;">36000</td>
			<td>catalog number for PTK reagent kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Serine/Threonine Kinase (STK) Array Profiling chip</td>
			<td style="width:184px;">PamGene</td>
			<td>87102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">STK kinase buffer</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;">32205</td>
			<td>catalog number for STK reagent kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">STK Primary Antibody Mix (DMAB tube)</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;">32205</td>
			<td>catalog number for STK reagent kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">FITC-conjugated Secondary Antibody</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;">32203</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">STK-MM1 tube (100X BSA)</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;">32205</td>
			<td>catalog number for STK reagent kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">STK Antibody Buffer</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;">32205</td>
			<td>catalog number for STK reagent kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Equipment</td>
			<td style="width:184px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">#11 Blades, sterile</td>
			<td style="width:184px;">Fisher</td>
			<td style="width:119px;">3120030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">#3 scalpel handles, sterile</td>
			<td style="width:184px;">Fisher</td>
			<td style="width:119px;">08-913-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">100mm glass Petri dishes</td>
			<td style="width:184px;">Fisher</td>
			<td style="width:119px;">08-748D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Semicurved forceps</td>
			<td style="width:184px;">Fisher</td>
			<td style="width:119px;">12-460-318</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Trypsinizing flask</td>
			<td style="width:184px;">Fisher</td>
			<td style="width:119px;">10-042-12B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Magnetic stirrer</td>
			<td style="width:184px;">Fisher</td>
			<td style="width:119px;">14-490-200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">3/4&#34; stir bar</td>
			<td style="width:184px;">Fisher</td>
			<td>14-512-125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">B-D cell strainer&#160;</td>
			<td style="width:184px;">Fisher</td>
			<td style="width:119px;">#352340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">B-D 50ml Centrifuge tube</td>
			<td style="width:184px;">Fisher</td>
			<td>#352098</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">PamStation 12</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">BioNavigator 6.0 kinomic analysis software&#160;</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Evolve Kinase Assay Software</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">UpKin App software (upstream kinase prediction)</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">gentleMACS Dissociator</td>
			<td style="width:184px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-093-235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Rotary Cell Culture System (RCCS)</td>
			<td style="width:184px;">Synthecon</td>
			<td style="width:119px;">RCCS-D</td>
			<td>with 10 mL disposable HARV</td>
		</tr>
	</tbody>
</table>

generation of microtumors using 3d human biogel culture system and patient-derived glioblastoma cells for kinomic profiling and drug response testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Nous avons montré que le 3D système de culture de Biogel soutient la croissance à long terme et la fonction des types cellulaires multiples. Dans ce projet de collaboration, xenolines de GBM patients dérivés (PDX) sont utilisés pour produire des centaines de microtumeurs. Les cellules dissociées (3 x 10 5) ou des neurosphères (40 - 50) ont été incorporés dans des perles de biogel (2 mm) et après gélification rapide , ils sont mis en culture dans un bioréacteur pe...

Watch this Scientific Journal Video about Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing at JoVE.

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

Génération de microtumeurs Utilisation 3D Biogel humain Système de culture et les cellules de glioblastome patients dérivés pour Kinomic Profilage et Testing Réponse Drug

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

The overall goal of this patient derived glioblastoma 3D microtumor culture system is to enable high-throughput drug testing with a corresponding molecular profiling assay, such as kinomic testing. This method can help answer key questions in the cancer field, such as, why do certain therapies work well in one patient's tumor but not in another? The main advantage of this technique is that it provides a miniature yet realistic tumor culture system suitable for therapy testing.Generally, individuals new to this method will struggle because handling the patient derived xenograft cells is not like handling long established, immortalized tumor cells. This is due to their fragility and sensitivity to changing culture conditions. Visual demonstration of this method is critical, as the microtumor generation steps are difficult to learn through description only, due to their unique and complex method of production.Demonstrating the tumor disaggregation procedure will be Catherine Langford, the senior technician and lab manager in my laboratory. Demonstrating the microtumor generation and drug dosing procedures will be Rachael Shevin, a technician in my lab. Using forceps, blunt dissect to gently free the tumor mass from the peritoneal wall and the skin, and transfer the tumor mass to a sterile glass Petri dish.Then, use a sterile scalpel with a number 11 blade and sterile semi-curved forceps to debride the tumor tissue of any necrotic tissue and membranous host connective tissue. Wash the cleaned tumors with sterile RPMI three to five times to remove the excess blood, gently tilting the dish between rinses to remove the wash. After the final wash, use two number 11 scalpel blades to finely mince the tumors.Then, use sterile semi-curved forceps to transfer the tumors into a dissociation tube containing enzyme solution. Place the tube into a cellular dissociator, and select the Tumor 2 program. Click Start"to run the dissociator for 37 seconds.Then transfer the tube to a rotator and place the rotator in a 37 degree Celsius incubator for 40 minutes. At the end of the incubation, place the tube back on the dissociator and run the Tumor 3 program for 37 seconds. At the end of the program, centrifuge the cells and re-suspend the pellet in 10 milliliters of serum free RPMI with gentle mixing.Next, slowly load the cells onto a 40 micron cell strainer in a new 50 milliliter tube, and allow the suspension to drip through the filter by gravity. Gently lift the tab of the cell strainer in between each new addition of cells, breaking the vacuum between the strainer and the tube to allow the suspended cells to pass freely through the mesh. When all of the cells have been filtered, spin down the single cell suspension and re-suspend the pellet in 10 milliliters of complete Neurobasal Medium.Then, determine the number of viable cells by trypan blue exclusion, and place the cells on ice. To generate the microtumors, spin down the dissociated patient derived glioblastoma xenoline cells and re-suspend the pellet at 50, 000 cells per two microliters of FBS. Next, dilute the cells in ice cold high density human biogel at a one to four cell to biogel ratio.Then, use an electronic multichannel pipette to dispense 10 microliters of cell mixture per pin onto a 96 pin steel plate with hydrophobic coating. It's important to prevent bubbles when mixing the cells, medium, and biogel, while still working quickly and efficiently to generate the microtumors before the human biogel begins to solidify on the pins. When all of the microtumors have been plated, transfer the 3D tumors into a tissue culture incubator for 20 minutes to gelate the tumor beads.At the end of the incubation, transfer the microtumors to a 10 centimeter culture dish containing complete Neurobasal Medium and return the tumors to the incubator. After one to two days, use a wide mouth dispensing pipette to transfer the microtumors into the wells of a 96 well culture plate, containing 50 microliters of Neurobasal Medium per well, and add 50 microliters of the appropriate dosing solution to each well. Maintain the treated microtumors in the tissue culture incubator for one to 14 days, feeding the tumors twice weekly with a fresh medium and drug solution.To evaluate the microtumor cell morphology, on days one, seven, and fourteen of culture, label the microtumors with one micromolar of freshly diluted Calcein-AM per well in the tissue culture incubator for 20 minutes. Then, image the tumors by flourescence microscopy at two, four, and 10x magnifications. To assess the microtumor growth, and days one, seven, and 14, treat the cultures with 20 microliters of freshly prepared MTT for two hours in the tissue culture incubator.At the end of the incubation, lyse the tumors with freshly prepared, 10%SDS lysis buffer at a one to one ratio to the culture well volumes, and seal the plate. Then incubate the microtumors in the lysis buffer overnight at 37 degrees Celsius, and read the absorbance at 570 nanometers on a multi-plate reader the next morning. Through live cell imaging Calcein-AM as just demonstrated, the cell growth and viability of the microtumors, as well as the effects of the drug therapies on these functions, can be evaluated.As observed, untreated microtumors and microtumors with a known resistance to the experimental drug treatment display a continuous growth pattern over a two week culture period. These TMZ sensitive tumor cells, however, demonstrate a reduced cell growth by the end of the culture in response to the highest concentration of TMZ treatment by MTT assay. Interestingly, kinase signaling in TMZ resistant microtumors reveals a number of peptides with significantly altered intensities after the 10 micromolar TMZ treatment.Indeed, in this representative experiment, analysis of these altered kinomic profiles using upstream kinase prediction software identified specific alterations in SYK, LCK, and CTK tyrosine kinase and a number of serine/threonine kinase intensities, suggesting potential novel therapeutic targets that may help overcome tumor treatment resistance. Once mastered, the tumor disaggregation can be completed in two hours if it is performed properly. The microtumor generation and drug dosing techniques can be completed in three hours total if they are performed properly.In the future, this model system could be used as a proband style avatar system, in which a patient's tumor is molecularly matched to an existing patient derived microtumor library to inform the clinician of the best choice of therapy.

Research

JoVE Journal

Medicine

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE

Identification et isolement des cellules ne se divisant lentement dans le glioblastome humain Utilisation carboxy fluorescéine succinimidyl ester (CFSE)

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic ...

Recherche

Médecine

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Différenciation isolement, caractérisation et comparative de l&#39;homme dentaires cellules souches de pulpe Dérivé de dents permanentes à l&#39;aide de deux méthodes différentes

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Patient Culture cellulaire dérivée et isolement de CD133<sup&gt; +</sup&gt; Putatifs cellules souches du cancer du mélanome

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Génération de cancer de la prostate des patients Dérivé modèles de xénogreffe de cellules tumorales circulantes

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Une tumeur au cerveau / organotypiques Slice système de co-culture pour l&#39;étude des tumeurs Microenvironnement et ciblée Thérapies de drogue

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...

Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells

Génération d&#39;un humanisée foie de souris Utilisation hépatique humaine Cellules souches

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in ...

Drug Repurposing Hypothesis Generation Using the "RE:fine Drugs" System

Système: Drug Repurposing Hypothesis Generation Utilisation de la &quot;Drogues fines RE&quot;

The promise of drug repurposing is that existing drugs may be used for new disease indications in order to curb the high costs and time for approval. The ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Forskoline induite par Gonflement dans Intestinal organites: Un<em&gt; In vitro</em&gt; Essai pour évaluer la réponse des médicaments dans les patients atteints de fibrose kystique

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Assessment of the Anticoagulant and Anti-inflammatory Properties of Endothelial Cells Using 3D Cell Culture and Non-anticoagulated Whole Blood

Évaluation des propriétés anticoagulantes et anti-inflammatoires de cellules endothéliales à l’aide de la Culture cellulaire 3D et sang total Non-anticoagulant

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood ...

Muscle Imbalances: Testing and Training Functional Eccentric Hamstring Strength in Athletic Populations

Les déséquilibres musculaires : Essais et formation fonctionnelle ischio-jambiers excentrique force athlétique populations

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite ...