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.

Ondersteund door NIH-subsidie ​​R21 (PI: C. Willey, CA185712-01), hersentumor SPORE award (PD: GY Gillespie, P20CA 151129-03) en SBIR contract (PI: R. Singh, N43CO-2013-00026).

OPMERKING: Alle xenograft therapie evaluaties werden uitgevoerd met behulp van een orthotope tumor model voor glioblastoma over een protocol door de Institutional Animal Care en gebruik Comite goedgekeurd. 1. Isolatie van de patiënt afgeleide GBM Xenograft Cells <ol><li> Bereiding van reagentia <ol><li> Opnieuw vormen collagenase-I in steriel water tot een concentratie van 5 mg / ml en steriel filter. Opslag in 1 ml porties bij -20 ° C (eindconcentratie 50 ug / ml in 100...

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Kritische stappen in het protocol voornamelijk betrekking op productie, en dosering van geneesmiddelen en onderhoud microtumor. Omdat de microtumor kralen zijn kwetsbaar en gemakkelijk gescheurd, is uiterste zorg nodig beide ontwikkelingsstadia van een test en onderhoud. Indien tijdens een van deze processen een fout optreedt, kan experimenteel interpretatie worden aangetast, waardoor verlenging of onnodige herhaling van experimenten of zelfs van gegevenssets. Wijzigingen en het oplossen van...

De meest voorkomende primaire intracraniële kwaadaardige hersentumoren zijn rang III astrocytomen en graad IV glioblastoma multiforme (glioblastoma of GBM). Deze tumoren bieden slechte prognose met een mediane één-jaars overleving tussen 12-15 maanden met de huidige therapieën voor GBM in de VS 1-3. Multimodaliteit behandelingen omvatten chirurgie, bestraling en chemotherapie waaronder temozolomide (TMZ) en-kinase gerichte agenten. Kinase signalering is vaak verstoord bij GBM, waaronder subgroepen van tumoren met amplificatie of activerende mutaties in het epidermale groeifactorreceptor (EGFR), verhogingen Platelet Derived Growth Factor Receptor (PDGFR) signalering, verhoogde fosfatidyl-inositol-3-kinase (PI3K) en tumor ondersteunen angiogene signalering door middel Vasculaire Endotheliale groei Factor receptor (VEGFR) en andere kinase pathways gedreven 4-6. Huidige in vitro en in vivo modellen vaak verliezen deze representatieve veranderingen &lt;sup&gt; 7. Daarnaast heeft genetische profielen niet aangeboden de verwachte voordelen kan het feit weerspiegelen dat genetische en epigenetische veranderingen wijzigingen niet altijd voorspellen op het niveau van eiwit activiteit, waar de meeste kinase richtende middelen direct werken, en wanneer therapieën met andere werkingsmechanismen kunnen fungeren indirect. De traditionele geïmmortaliseerde cellijn die kan worden gepasseerd eindeloos is al lang de standaard voor drug tests om goede houdbaarheid en reproduceerbaarheid. Echter, dit model lijdt aan een hoge nutriënt (en kunstmatige) groei omgeving die kiest voor het snel groeiende cellen die sterk afwijken van de oorspronkelijke tumor. Als zodanig is er aanzienlijke belangstelling voor de ontwikkeling realistischer modelsystemen die een complexere tumor biologisch systeem reflecteren zoals aanwezig in de patiënt is. Tumorxenograften direct ontwikkeld van een primaire tumor gegroeid in muizen ( &quot;xenoline,&quot; patiënt afgeleide xenograft of PDX) provide een reflectief modelsysteem, met name in de context van kanker therapeutica, zoals zij geacht worden klinisch succes meer betrouwbare voorspelling. 8 Ondanks een reflecterende biologie, deze modellen zijn duur en zijn moeilijk vast te stellen en te onderhouden. Bovendien zijn zij niet vatbaar zijn voor high-throughput studies. De noodzaak om biologische modellen die een betere weerspiegeling van moleculaire veranderingen in de primaire tumoren, en naar het profiel en test deze modellen met behulp van directe metingen van kinase-activiteit, niet surrogaat genetische merkers beter te ontwikkelen, is duidelijk. Het is algemeen bekend dat in tegenstelling tot tweedimensionale (2D) monolaag culturen, 3D of meercellige assay modellen kunnen meer fysiologisch bieden relevante eindpunten 9-11. Common 3D cultuur benaderingen betrekken-matrix gecoate microdragers en de vorming cel sferoïde. Bolvormige tumoren kunnen worden gegenereerd via cellulaire aggregatie met behulp van centrifugekolf, pHEMA bord en opknoping druppel technieken. Beperkingen voor teze benaderingen omvatten: de onmogelijkheid voor een aantal cellen om stabiele sferoïden, variabiliteit in de groei en uitdagingen met gemengde celtypen te vormen. Als alternatief, vele synthetische (hydrogel, polymeer) en dierlijke-afgeleide Engelbreth-Holm-Swarm (EHS) matrix van muis sarcomen, runder collageen) matrices zijn ontwikkeld voor 3D-cultuur bestudeert 12-14. Muis EHS matrix wordt uitgebreid gebruikt maar bekende celgroei en differentiatie bevorderen in vitro en in vivo 15. Om 3D tumorbiologie repliceren, werd een menselijke biomatrix ontwikkeld door Dr. Raj Singh et al. 16. De natuurlijke, groeifactor-vrij mens Biogel maakt 3D-cultuur steigers (kralen, schijven), die op lange termijn de teelt van meerdere celtypen te ondersteunen. Een reeks 3D menselijke Biogel cultuur ontwerpen worden vastgesteld voor het bestuderen van tumorgroei, adhesie, angiogenese en invasie eigenschappen. Voordelen en eigenschappen van menselijke Biogel vergeleken met gewonemuis EHS gels worden samengevat in tabel 1 en tabel 2. <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Bron: </td><td> Human Amnions (Pooled weefsel) Pathogeen-vrij, IRB-vrijgestelde / goedgekeurde </td></tr><tr><td> ECM natuur: </td><td> Niet-gedenatureerde Biogel (GLP-productie) </td></tr><tr><td> sleutel componenten: </td><td> Col-I (38%), laminine (22%), Col-IV (20%), Col-III (7%), entactine en HSPG (&lt;3%) </td></tr><tr><td> GF-vrij: </td><td> Undetectable EGF, FGF, TGF, VEGF, PDGF (Non-angiogene niet-toxisch) </td></tr></tbody></table> Tabel 1: Eigenschappen van Human Biogel als Vergeleken met Common EHS Gels. <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page = &quot;always&quot;&gt; <tbody><tr><td> Human Biogel </td><td> EHS gels </td></tr><tr><td> Natuurlijke menselijke matrix </td><td> Opgeloste muis matrix </td></tr><tr><td> Gecontroleerde groei &amp; differentiatie cel </td><td> Kan bevorderen celgroei en differentiatie </td></tr><tr><td> Fysiologische genexpressie </td><td> Variabele genexpressie </td></tr><tr><td> 3D weefsel-achtige cultuur model </td><td> -Plaat gebaseerde cultuur model </td></tr></tbody></table> Tabel 2: Voordelen van Human Biogel als Vergeleken met Common EHS Gels.

<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.

We hebben laten zien dat 3D Biogel cultuur systeem ondersteunt groei op lange termijn en de functie van meerdere celtypen. In dit samenwerkingsproject, zijn patiënt afgeleid GBM xenolines (PDX) gebruikt voor de productie van honderden microtumors. Gedissocieerde cellen (3 x 10 5) of neurosferen (40 - 50) zijn ingebed in Biogel parels (2 mm) en na snelle gelering ze gekweekt in een NB-medium gevuld custom bioreactor. Cellulaire levensvatbaarheid (calceïne-AM), groeiprofiel (M...

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.

Genereren van Microtumors Met behulp van 3D Human Biogel Cultuur System en Patient afgeleide glioblastoma Cellen voor Kinomic Profiling and Drug Response Testen

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

Identificatie en isolatie van Slow-delende cellen in Human Glioblastoma Met behulp van Carboxy fluoresceïne succinimidylester (CFSE)

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

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

Isolatie, karakterisering en vergelijkende differentiatie van humane Dental Pulp stamcellen uit permanente tanden met behulp van twee verschillende methoden

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

Patiënt Derived Cell Cultuur en isolatie van CD133<sup&gt; +</sup&gt; Vermeende Cancer Stem Cells van Melanoom

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

Generatie van prostaatkanker Patiënt Afgeleid xenotransplantaatmodellen van circulerende tumorcellen

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

Een hersentumor / organotypische Slice Co-cultuur voor het bestuderen tumor micro en Gerichte Drug therapieën

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

Genereren van een gehumaniseerd muis lever met behulp van menselijke lever stamcellen

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

Drug herbestemming Hypothese Generation Met behulp van de &quot;RE: fine Drugs&quot; System

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-geïnduceerde zwelling in Intestinal Organoids: An<em&gt; In Vitro</em&gt; Assay voor het beoordelen van de Drug Response in patiënten met cystische fibrose

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

Beoordeling van de anticoagulatie en anti-inflammatoire eigenschappen van endotheliale cellen met behulp van 3D-celkweek en niet-vertonen volbloed

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

Spier onevenwichtigheden: Testen en functionele excentrieke Hamstring sterkte in atletische populaties opleiding

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