（：C.ウィリー、CA185712-01 PI）、脳腫瘍SPORE賞（PD：GYギレスピー、P20CA 151129から03）とSBIR契約（PI：R.シン、N43CO-2013から00026）NIH R21グラントによってサポートされています。

<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>

R.S. and R.S.S. are employees of Vivo Biosciences. J.C.A. is partially funded by the UAB Kinome Core.

プロトコル内の重要なステップは、主に世代をmicrotumorに関連するだけでなく、薬剤投与とメンテナンス。 microtumorビーズが壊れやすく、簡単に引き裂かれたので、細心の注意をアッセイおよびメンテナンスの発達段階の両方で必要とされます。エラーがこれらのプロセスのいずれかの間に発生した場合は、実験的な解釈は、拡張機能や実験の不必要な繰り返しやデータのさえ排除を引き起こ?...

最も一般的な原発頭蓋内悪性脳腫瘍は、グレードIII星状細胞腫およびグレードIVの多形性膠芽腫 （神経膠芽腫またはGBM）です。米国1-3でGBMに対する現在の治療と15ヶ月-これらの腫瘍は、12の間の中央値1年生存率と予後不良を提供しています。マルチモダリティ療法は、テモゾロミド（TMZ）およびキナーゼを標的とする薬剤を含む外科手術、放射線、および化学療法が含まれます。キナーゼシグナル伝達は、頻繁に上皮成長因子受容体（EGFR）、血小板由来増殖因子受容体（PDGFR）の増加、シグナリング、増加したホスファチジルイノシトール3キナーゼ（PI3K）とにおける増幅を有する腫瘍のサブセットまたは活性化変異を含む、GBMで調節不全されます血管内皮増殖因子受容体（VEGFR）、ならびに他のキ ​​ナーゼ駆動経路4-6を介して血管新生シグナル伝達を支援する腫瘍。 in vitroおよびin vivoモデルで現在は頻繁にこれらの代表的な変化を失います&lt;SUP&gt; 7。さらに、遺伝的プロファイリングは、遺伝的およびエピジェネティックな変化は常に標的剤、ほとんどのキナーゼが直接作用するタンパク質活性のレベルで変化を予測せず、他の作用機序を持つ治療法がどこに作用することができるという事実を反映している可能性が予想される利益を提供していません間接的に。 無限に継代することができる伝統的な不死化細胞株は、長いによるメンテナンスや再現が容易に薬物検査のための標準となっています。しかし、このモデルは、高速、元の腫瘍から大きく異なる細胞を増殖させるための選択の高い栄養素（人工）成長環境に苦しんでいます。このように、患者に存在するように、より複雑な腫瘍生物系を反映する、より現実的なモデル系を開発することにかなりの関心が集まっています。腫瘍異種移植片は、マウスで増殖させた原発腫瘍（「xenoline、「患者由来の異種移植片またはPDX）専らから直接開発しますそれらは、より確実に臨床的成功を予測することが8以上の反射生物学にもかかわらず感じられるようにデは、特に癌治療の設定で複数の反射モデル系は、これらのモデルは、高価であり、確立し、維持するのが困難です。さらに、それらは、ハイスループット試験に適していません。より良い、より正確に原発腫瘍の分子変化を反映して、遺伝子マーカーの代理、キナーゼ活性の直接的尺度を使用して、これらのモデルをしないプロファイルおよびテストするために生物学的モデルを開発する必要性が、明らかです。 よく、二次元（2D）単層培養とは異なり、3次元または多分析モデルは、より生理学的に関連するエンドポイント9-11を提供することができることが認識されます。一般的な3D培養手法は、マトリックスコートしたマイクロキャリアおよび細胞スフェロイド形成を伴います。腫瘍スフェロイドは、スピナーフラスコのpHEMA板と懸滴技術を使用して細胞凝集を介して生成することができます。トンの制限事項HESEアプローチは、次のとおりです。安定したスフェロイドを形成するためにいくつかの細胞のための不能、混合細胞型と成長と課題の変動を。あるいは、合成（ハイドロゲル、ポリマー）およびマウス肉腫から動物由来エンゲル・ホルム-スウォーム（EHS）行列多く、ウシコラーゲン）行列は3D培養のために開発されてきた12-14を研究しています。マウスEHSマトリックスは、広く使用されるが、in vitroおよびin vivo 15細胞の増殖および分化を促進することが知られています。 3D腫瘍生物学を複製するためには、人間のバイオマトリックスシステムは、博士のRaj Singh氏らによって開発された。16。 天然の、成長因子を含まないヒトのバイオゲルは、複数の細胞型の長期培養をサポートする3D培養の足場（ビーズ、ディスク）を可能にします。 3Dヒトバイオゲル培養設計のシリーズは、腫瘍増殖、接着、血管新生および浸潤特性を研究するための確立されています。利点と人間のバイオゲルのプロパティ共通に比べて、マウスEHSゲルは、表1および表2にまとめます。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> ソース： </td><td> 人間Amnions（プールされた組織） 病原体を含まない、IRB-免除/承認</td></tr><tr><td> ECMの性質： </td><td> 非変性バイオゲル （GLP-生産） </td></tr><tr><td> キー コンポーネント： </td><td> COL-I（38％）、ラミニン（22％）、COL-IV（20％）、COL-III（7％）、エンタクチンおよびHSPG（&lt;3％） </td></tr><tr><td> GF-フリー： </td><td> 検出不可能 EGF、FGF、TGF、VEGF、PDGF（非血管新生性、非毒性） </td></tr></tbody></table> 表1：一般的なEHSゲルと比較して、ヒトバイオゲルのプロパティ。 <table border="1" fo:keep-together.within-page="1" fo:keep-wiTH-next.within-ページ= &quot;常に&quot;&gt; <tbody><tr><td> 人間バイオゲル </td><td> EHSゲル </td></tr><tr><td>人間の自然な行列</td><td>再構成されたマウスの行列</td></tr><tr><td>制御された細胞増殖＆分化</td><td>細胞増殖＆分化を促進することができます</td></tr><tr><td>生理学的遺伝子発現</td><td>可変遺伝子発現</td></tr><tr><td> 3D組織様培養モデル</td><td>プレートベース培養モデル</td></tr></tbody></table> 表2：一般的なEHSゲルと比較して、ヒトバイオゲルの利点。

<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.

我々は、3Dバイオゲル培養系は、複数の細胞型の長期的成長と機能をサポートしていることを示しています。この共同プロジェクトでは、患者由来のGBM xenolines（PDX）は微小腫瘍の数百を製造するために使用されています。解離した細胞（3×10 5）またはニューロスフェア（40 - 50）バイオゲルビーズ（2mm）に包埋し、迅速なゲル化した後、それらはNB-媒体充填カ...

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.

Kinomicプロファイリングと薬物反応検査のための3D人間バイオゲル培養システムと患者由来の神経膠芽腫細胞を用いた微小腫瘍の発生

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

generation-microtumors-using-3d-human-biogel-culture-system-patient

Research

JoVE Journal

Medicine

9.0K ビュー.  University of Alabama at Birmingham. 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. 

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

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

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic strategies1. To overcome the issue of tumor heterogeneity, it is essential to develop assays and tools enabling phenotypic, (epi)genetic and functional identification and characterization of tumor subpopulations that drive specific disease pathologies and represent clinically relevant targets. It is now well established that tumors exhibit distinct sub-fractions of cells with different frequencies of cell division, and that the functional criteria of being slow cycling is positively associated with tumor formation ability in several cancers including those of the brain, breast, skin and pancreas as well as leukemia2-8. The fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) has been used for tracking the division frequency of cells in vitro and in vivo in blood-borne tumors and solid tumors such as glioblastoma2,7,8. The cell-permeant non-fluorescent pro-drug of CFSE is converted by intracellular esterases into a fluorescent compound, which is retained within cells by covalently binding to proteins through reaction of its succinimidyl moiety with intracellular amine groups to form stable amide bonds9. The fluorescent dye is equally distributed between daughter cells upon divisions, leading to the halving of the fluorescence intensity with every cell division. This enables tracking of cell cycle frequency up to eight to ten rounds of division10. CFSE retention capacity was used with brain tumor cells to identify and isolate a slow cycling subpopulation (top 5% dye-retaining cells) demonstrated to be enriched in cancer stem cell activity2. 
This protocol describes the technique of staining cells with CFSE and the isolation of individual populations within a culture of human glioblastoma (GBM)-derived cells possessing differing division rates using flow cytometry2. The technique has served to identify and isolate a brain tumor slow-cycling population of cells by virtue of their ability to retain the CFSE labeling.

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

This video protocol demonstrates the application of the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) for the identification and separation of different sub-populations of cells in human glioblastoma based on frequency of cell division.

カルボキシフルオレセインスクシンイミジルエステル（CFSE）を用いてヒト神経膠芽腫のスロー非分裂細胞の同定および単離

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

医学

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

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

単離、特徴付けと二つの異なる方法を使用して永久歯に由来するヒト歯髄幹細胞の分化の比較

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

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

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

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

CD133の患者由来の細胞培養および分離<sup&gt; +</supメラノーマから&gt;推定されるがん幹細胞

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

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

臨床応用のためのヒト羊膜上皮細胞の単離、凍結保存と文化

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

病気のモデリングのための筋生検からの患者由来の細胞株の単離と不死

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

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

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

前立腺癌患者の発生は、循環腫瘍細胞からの異種移植モデルを派生します

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

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

腫瘍微小環境と標的薬物療法研究のための脳の腫瘍/器官スライス共培養系

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

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

ヒト下垂体腺腫細胞の三次元アルギン酸ビーズ文化

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in which the liver has been repopulated with functional human hepatocytes, could serve as a useful tool for investigating human hepatic cell biology, drug metabolism, and other preclinical applications. One of the key factors required for successful transplantation of human hepatocytes into mice is the elimination of the endogenous hepatocytes to prevent competition with the human cells and provide a suitable space and microenvironment for promoting human donor cell expansion and differentiation. To date, two major liver injury mouse models utilizing fumarylacetoacetate hydrolase (Fah) and uroplasminogen activator (uPA) mice have been established. However, Fah mice are used mainly with mature hepatocytes and the application of the uPA model is limited by decreased breeding. To overcome these limitations, Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice were used for in vivo differentiation of immature human hepatocytes and humanized liver generation. Human hepatic stem cells (HpSCs) successfully repopulated the livers of Alb-TRECK/SCID mice that had developed lethal fulminant hepatic failure following diphtheria toxin (DT) treatment. This model of a humanized liver in Alb-TRECK/SCID mice will have functional applications in studies involving drug metabolism and drug-drug interactions and will promote other in vivo and in vitro studies.

Here, we present a novel humanized mouse liver model generated in Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice following the transplantation of immature and expandable human hepatic stem cells.

ヒト肝幹細胞を用いたヒト化マウス肝臓の生成

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

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 goal of computational methods for drug repurposing is to enable solutions for safer, cheaper and faster drug discovery. Towards this end, we developed a novel method that integrates genetic and clinical phenotype data from large-scale GWAS and PheWAS studies with detailed drug information on the concept of transitive Drug-Gene-Disease triads. We created "RE:fine Drugs," a freely available, interactive dashboard that automates gene, disease and drug-based searches to identify drug repurposing candidates. This web-based tool supports a user-friendly interface that includes an array of advanced search and export options. Results can be prioritized in a variety of ways, including but not limited to, biomedical literature support, strength and statistical significance of GWAS and/or PheWAS associations, disease indications and molecular drug targets. Here we provide a protocol that illustrates the functionalities available in the "RE:fine Drugs" system and explores the different advanced options through a case study.

Here we describe a protocol using the web-based drug repurposing hypothesis generation tool: "RE:fine Drugs." This protocol can be modified to a user's preferences at the level of the query type (gene, drug or disease) and/or the range of available advanced options.

薬物転用仮説生成の使用」RE：罰金薬「システム

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 ...