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.

, Beyin Tümörü SPORE ödülü (PD: GY Gillespie, P20CA 151129-03) ve SBIR sözleşmesi (PI: R. Singh, N43CO-2013-00026): NIH R21 hibe (C. Willey, CA185712-01 PI) tarafından desteklenmektedir.

NOT: Tüm ksenograft tedavi değerlendirmeler Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından onaylanmış bir protokole glioblastoma için ortotopik tümör modeli kullanılarak yapılmıştır. Hasta türetilen GBM Xenograftlarında Hücreleri 1. İzolasyon <ol><li> Reaktiflerin hazırlanması <ol><li> 5 mg / ml steril filtre bir konsantrasyona, steril su içinde kolajenaz-re-oluşturmaktadır. -20 ° C&#39;de 1 ml&#39;lik numuneler halinde saklayın (nihai konsan...

<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>

protokolü içinde kritik adımlar ağırlıklı nesil yanı sıra ilaç dozajı ve bakım microtumor ilgilidir. microtumor boncuk kırılgan ve kolayca yırtılmış olduğu için, son derece dikkatli bir tahlil ve bakım gelişim evreleri, hem de ihtiyaç vardır. Bir hata Bu işlemlerden birini sırasında oluşursa, deneysel yorumlama uzantısı veya deneylerin gereksiz tekrarını hatta veri dışlanmasını neden tehlikeye girebilir. Değişiklikler ve sorun giderme, özellikle microtu...

En sık rastlanan primer intrakranial malign beyin tümörleri evre III astrositom ve Evre IV glioblastoma multiforme (glioblastoma veya GBM) bulunmaktadır. ABD 1-3 GBM için geçerli tedavilerle 15 ay - Bu tümörler 12 arasında ortalama bir yıllık sağkalım yoksul prognoz sunuyoruz. Çok modelli tedaviler temozolomid (TMZ) ve kinaz hedefli ajanlar da dahil olmak üzere cerrahi, radyasyon ve kemoterapi yer alıyor. Kinaz sinyal sık Epidermal Büyüme Faktörü reseptörü (EGFR), Trombosit Türevli Büyüme Faktörü Alıcısının (PDGFR) artışlar sinyal, artan Fosfatidil-inositol-3 kinaz (PI3K) ve amplifikasyon ile tümör alt-veya aktive mutasyonlar da dahil olmak üzere, GBM&#39;de düzensiz olan Vasküler Endotel Büyüme Faktörü Alıcısının (VEGFR) hem de diğer kinaz tahrik yollar 4-6 ile anjiyojenik sinyal destekleyici tümörü. Vitro ve in vivo modelleri sık sık bu temsili değişiklikleri kaybetmek Güncel &lt;sup&gt; 7. Buna ek olarak, genetik profilleme genetik ve epigenetik değişiklikler her zaman hedefleme ajanları çoğu kinaz doğrudan hareket protein aktivite düzeyi, değişiklik tahmin yok ve eylem diğer mekanizmalar ile tedaviler nerede hareket edebilirler gerçeğini yansıtıyor olabilir beklenen faydalar teklif yok dolaylı olarak. sonsuza pasajlandı edilebilir geleneksel ölümsüzleştirdi hücre hattı uzun nedeniyle bakım ve tekrarlanabilirlik kendi kolaylığı uyuşturucu testi için standart olmuştur. Ancak, bu model hızlı orijinal tümörden önemli ölçüde farklıdır hücrelerin büyümesi için seçer yüksek besin (ve yapay) bir büyüme ortamına muzdarip. Bunun gibi, hastanın mevcut olduğu gibi daha karmaşık tümör biyolojik sistemi yansıtacak daha gerçekçi bir model sistemlerinin geliştirilmesinde önemli bir ilgi olmuştur. Tümör ksenogrefleri farelerde yetişen bir primer tümörün ( &quot;xenoline,&quot; hasta-türevi xenograftlarının veya PDX) yazarlara doğrudan geliştirilenOnlar daha güvenilir klinik başarısını tahmin etmek. 8 daha yansıtıcı biyoloji rağmen keçe gibi de özellikle kanser tedavi ortamında bir daha yansıtıcı bir model sistem, bu modeller pahalıdır ve kurmak ve sürdürmek zordur. Ayrıca, yüksek verimli çalışmalar için uygun değildir. Gerek daha daha doğru genetik belirteçleri vekil değil, birincil tümörlerde moleküler değişikliklerin yansıtmak ve profil ve kinaz aktivitesinin doğrudan önlemler kullanarak bu modelleri test biyolojik modellerin geliştirilmesi, açıktır. Iki-boyutlu (2B) tek tabakalı kültürler farklı olarak, 3 boyutlu veya çok-hücreli deney modelleri daha fazla fizyolojik olarak ilgili son noktaları 9-11 sağlayabilir kabul edilmektedir. Ortak 3D kültür matris kaplı mikrotaşıyıcılar ve hücre sfero oluşumunu içeren yaklaşımlar. Tümör Sferoidler spinner şişesi, PHEMA plaka kullanarak ve bırak teknikleri asılı hücresel toplama yoluyla elde edilebilir. t Sınırlamalarhese yaklaşımlar şunlardır: bazı hücreler karışık hücre tipleri ile istikrarlı parçacıklarının, değişkenliği büyüme ve zorlukları oluşturmak için yetersizlik. Seçenek olarak ise, bir çok sentetik (hidrojel polimer) ve Engelbreth-Holm-Swarm matrisler 3D kültür için geliştirilmiş olan fare sarkom den (EHS) matris, bovin kolajen) 12-14 çalışmalar hayvan türevi. Farenin EHS matrisi yaygın olarak kullanılan, ancak in vitro ve in vivo 15 hücre büyümesini ve farklılaşmasını teşvik ettiği bilinir. 3D tümör biyolojisi çoğaltmak için, bir insan biyomatris sistemi Dr. Raj Singh ve ark., 16 tarafından geliştirilmiştir. Doğal, büyüme faktörü içermeyen insan BioGel birden fazla hücre tipleri uzun süreli yetiştirme destek 3D kültür iskeleleri (boncuklar, diskler) izin verir. 3D insan BioGel kültür tasarımları bir dizi tümör büyümesi, yapışma, anjiyogenez ve işgali özelliklerini incelemek için kurulmuştur. Avantajları ve insan BIOGEL özellikleri ortak karşılaştırıldığındafare EHS jeller Tablo 1 ve Tablo 2&#39;de özetlenmiştir. <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Kaynak: </td><td> İnsan Amnions (toplanan doku) Patojen içermeyen, IRB muaf / onaylı </td></tr><tr><td> ECM doğa: </td><td> Sigara denatüre BIOGEL (GLP-üretim) </td></tr><tr><td> Anahtar bileşenleri: </td><td> Col-I (% 38), laminin (% 22), col-IV (% 20), col-III (% 7), entactin ve HSPG (&lt;% 3) </td></tr><tr><td> GF-serbest: </td><td> Saptanamaz EGF, FGF, TGF, VEGF, PDGF (Non-anjiyogenik, toksik olmayan) </td></tr></tbody></table> Tablo 1: Ortak EHS Gels karşılaştırıldığında İnsan BIOGEL özellikleri. <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page = &quot;always&quot;&gt; <tbody><tr><td> İnsan BIOGEL </td><td> EHS jeller </td></tr><tr><td> Doğal insan matris </td><td> Sulandırılarak fare matrisi </td></tr><tr><td> Kontrollü hücre büyümesi farklılaşma </td><td> hücre büyümesini ve farklılaşmasını teşvik edebilir </td></tr><tr><td> Fizyolojik gen ekspresyonu </td><td> Değişken gen sentezleme </td></tr><tr><td> 3D doku gibi kültür modeli </td><td> Plaka tabanlı kültürü modeli </td></tr></tbody></table> Tablo 2: Ortak EHS Gels karşılaştırıldığında İnsan BIOGEL avantajları.

<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.

Biz 3D BioGel kültür sistemi birden fazla hücre tiplerinin uzun vadeli büyüme ve fonksiyonunu destekler olduğunu göstermiştir. Bu ortak bir proje, hasta türetilmiş GBM xenolines (PDX) microtumors yüzlerce üretmek için kullanılır. Ayrılan hücreler (3 x 10 5) ya da Neurospheres (40-50) BioGel boncuklar (2 mm) içine gömüldü ve hızlı jelleşme sonra bir NB-ortam dolu özel biyoreaktör kültürlenir. Hücresel canlılığı (Calcein-AM), büyüme profili (MT...

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 Profil Yöntemi ve İlaç Tepki Testi için 3D İnsan BIOGEL Kültür Sisteminin Kullanılması Microtumors ve Hasta kaynaklı Glioblastom Hücreleri Üretimi

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

Karboksi Flöresein sukkinimidil ester (KAKE) Kullanarak İnsan Glioblastoma Yavaş-bölme Hücreleri Tanımlama ve İzolasyonu

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

Tıp

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

İnsan Diş Pulp Kök Hücre İzolasyonu, Karakterizasyonu ve Karşılaştırmalı Farklılaşma İki Farklı Yöntemler Kullanılarak Daimi Dişler türetilen

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

Hasta Türetilen Hücre Kültürü ve CD133 İzolasyonu<sup&gt; +</supMelanom dan&gt; sağlayan olası Kanser Kök Hücreleri

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

Prostat Kanseri Hasta Üretimi Tümör Hücreleri Sirkülasyon gelen Xenograftlarında Modeller Türetilmiş

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

İlaç Tedavileri tümörün mikro incelenmesi ve Hedeflenen A Beyin Tümörü / Organotipik Dilim Co-kültür Sistemi

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

İnsan Karaciğer Kök Hücreler kullanma Insanlaşmış Fare Karaciğer Üretimi

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

&quot;RE: İnce ilaçlar kullanılması&quot; İlaç repurposing Hipotez Üretim Sistemi

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

Forskolin kaynaklı bağırsak Organoids şişme: Bir<em&gt; İn Vitro</em&gt; Deney Kistik Fibrozis Hastalarında İlaç Tepki değerlendirilmesi için

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

Endotel hücreleri kullanarak 3D hücre kültürü ve sigara anticoagulated tam kan antikoagülan ve anti-enflamatuar özellikleri değerlendirilmesi

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

Kas dengesizlikleri: Test ve fonksiyonel eksantrik Hamstring gücü atletik nüfus eğitim

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