Yazarlar, Ulusal Doğa Bilimleri Vakfı Çin (31371390), devlet yüksek teknoloji geliştirme projesi (2014AA020702) programı ve programın Guangdong bilim ve teknoloji (2016B030231001) araştırma hibe destek kabul etmiş oluyorsunuz.

Tüm hayvan iş hayvan refahı Yasası, kurumsal yönergelere uygun olarak gerçekleştirilen ve kurumsal hayvan bakım ve kullanım Komitesi, Sun Yat-Sen'in Üniversitesi tarafından onaylanmıştır.
1. TEM hazırlanması
<ol><li>Fareler tarafından servikal çıkığı yürütmek ve steril cerrahi makas ve cımbız kullanarak dilleri kaldırın.</li>
	<li>Tongues yılında % 75 etanol 3 min için bırakın, sonra her dil bir 1,5 mL koymak Eppendorf (EP) tüp ile 1 mL 10 mM steril fosfat tamponlu çözüm (PBS). Not: PBS konsantrasyonu aşağıdaki adımlarda bu adımda konsantrasyon olarak aynıdır.</li>
	<li>Lizis donma çözülme tarafından hücre:-80 ° c için 1 h EP tüpler dilleri donma ve 3 kür için dilleri için 45 dakika oda sıcaklığında erimek.</li>
	<li>Cerrahi sütür cerrahi iğne kullanarak bir parça üzerine her dil yüklemek ve steril folyo küçük bir parça ile dikiş sonu sarın. % 75 etanol steril koşullarda içeren bir 3,5 cm veya 6 cm kültür tabağına işlemi. Not: Uygun da cerrahi sütür her parçası yaklaşık 20 cm uzunluğunda ve 0,3 cm2 hakkında (1 cm × 0,3 cm) folyo her parçasının uygun boyutudur. Dil folyo yüklü olmalıdır.</li>
	<li>Her dil 3 mL steril PBS ile 30 s. gerçekleştir steril koşullarda bu işlem için bir 3,5 cm veya 6 cm kültür tabak içinde yıkayın.</li>
	<li>Dilleri Ultrasaf Su ile yıkamak: ampicilin geniş ağız şişe içine steril Ultrasaf Su 250 mL ile 90 µg/mL nihai bir konsantrasyon için ekleyin. Dilleri içeren bir heyecan bar şişe içine koymak. Şişe kapağı şişe dışında kalan dikiş parçası ile sıkın. Bu işlemi steril koşullarda gerçekleştirmek. Not: en fazla 5 dilleri dikiş twining söz konusu aynı şişe içine koyabilirsiniz. Folyo şişe dil kapalı kaymasını önlemek için dikiş sonu altındadır. Dilleri şişe içinde kalan dikiş uzunluğunu ayarlayarak şişenin alttan 2 cm yüksekliğinde yerleştirilmelidir. Bu Not için adımları 1.8, 1.10, 1.12 ve 1,16 da.</li>
	<li>Manyetik karıştırıcı 12 h için şişe koymak.</li>
	<li>Dilleri NaCl ile yıkayın: ampicilin geniş ağız şişe içine steril 1 250 mL ile eklemek M NaCl 90 µg/mL nihai bir konsantrasyon için. Dilleri ve heyecan bar şişe içine taşıyın. Şişe kapağı şişe dışında kalan dikiş parçası ile sıkın. Bu işlemi steril koşullarda gerçekleştirmek.</li>
	<li>Manyetik karıştırıcı 24 h için şişe koymak.</li>
	<li>Hücre lizis Triton X-100 tarafından: 90 µg/mL 250 mL steril % 2 ile son bir konsantrasyon geniş ağız şişe içine ampicilin eklemek PBS Triton X-100. Dilleri ve heyecan bar şişe içine taşıyın. Şişe kapağı şişe dışında kalan dikiş parçası ile sıkın. Bu işlemi steril koşullarda gerçekleştirmek.</li>
	<li>Manyetik karıştırıcı 48 h için şişe koymak.</li>
	<li>Dilleri CaCl2/MgCl2yıkama: ampicilin geniş ağız şişe içine steril 5 mM CaCl2/MgCl2 250 mL ile 90 µg/mL nihai bir konsantrasyon için ekleyin. Dilleri ve heyecan bar şişe içine taşıyın. Şişe kapağı şişe dışında kalan dikiş parçası ile sıkın. Bu işlemi steril koşullarda gerçekleştirmek.</li>
	<li>Manyetik karıştırıcı 24 h için şişe koymak.</li>
	<li>DNaz tarafından sindirim: Hank'in 1 mL dengeli tuz solüsyonu (HBSS) her EP tüp için ekleyin. DNaz HBSS sırasıyla 300 son bir konsantrasyon için eklemek µM. taşımak her dil tüp dışında dikiş parçası ile her EP tüpün içine. Bu işlemi steril koşullarda gerçekleştirmek. Not: yukarıdaki adımı şişelerde içinde kalır dikiş parçası da bu adımda EP tüp içinde kalır emin olun ve önceki adımları şişelerde dışında kalır dikiş parçası da bu adımda EP tüp dışında kalır emin olun.</li>
	<li>EP tüpler için 24 h 37 ° C'de dilleri kuluçkaya.</li>
	<li>Dilleri PBS ile yıkayın: ampicilin geniş ağız şişe içine steril PBS 250 mL ile 90 µg/mL nihai bir konsantrasyon için ekleyin. Dilleri ve heyecan bar şişe içine taşıyın. Şişe kapağı şişe dışında kalan dikiş parçası ile sıkın. Bu işlemi steril koşullarda gerçekleştirmek.</li>
	<li>Manyetik karıştırıcı 24 h için şişe koymak.</li>
	<li>Hazırlanan TEM 4 ° C'de steril PBS kadar kullanmak saklayın.</li>
</ol>2. TSCC üç boyutlu (3D) sulandırma
<ol><li>
Statik TSCC modeli İnşaat
	<ol>
<li>Tohum 1.0 x 106 tek TSCC hücreler (Cal27) 3.5 cm kültür çanak içine. Dulbecco'nın 3 mL kartal orta/F12 (DF12) içeren % 10 fetal Sığır serum (FBS), 90 µg/mL ampicilin ve 90 µg/mL sefaloridin modifiye ekleyin.</li>
		<li>Cal27 hücreleri 37 ° C'de 2-3 gün için kültür. Hücreleri en az % 60 cover emin olun alan çanak alt.</li>
		<li>TEM Cal27 monolayer kültür tabağına üzerine yükleyin.</li>
		<li>Çanak 28 gün boyunca bir CO2 kuluçka 37 ° C'de koyun.</li>
		<li>Kültür Orta hücre kültür işlemi sırasında her gün yenileyin. Kuluçka CO2 konsantrasyonu % 5 var.</li>
	</ol>
</li>
	<li>
Karıştırılmış TSCC modeli İnşaat
	<ol>
<li>
Kendi yaptığınız karıştırılmış minibioreactor hazırlanması
		<ol>
<li>Dalgıç 10 mL şırınga dan çıkar.</li>
			<li>Çubuk daha düşük terminal bir delik (1 cm çapında) kazmak ve delik bir heyecan bar yük.</li>
			<li>Bir delik (çapı 0.5 cm) geniş ağız şişe Plastik şişe kapağı merkezinde kazmak ve piston kolu ile kap koymak.</li>
			<li>50 mL santrifüj tüpü yarısı kesilmiş ve şişe kapağı dış tarafında kaynak.</li>
			<li>Eşyaları için çubuk çubuk eşyaları için bağlı balıkçılık satırlar ile sarma tarafından iliştirin. Not: en fazla 4 eşyaları bir çubuk için eklenebilir.</li>
			<li>Otoklav kullanmadan önce kendi kendine yapılan karmaşık. Not: değil basınçlı kap plastik geniş ağız şişe var. Yeni bir steril plastik geniş ağız şişe hücre kültürü çalışmalarının ise kullanın.</li>
		</ol>
</li>
	</ol>
</li>
	<li>
Dinamik hücre kültürü
	<ol>
<li>Tohum 1.0 x 106 tek Cal27 hücrelerde kendi kendine yapılan minibioreactor. %10 FBS, 90 µg/mL ampicilin ve 90 µg/mL sefaloridin içeren DF12 orta 150 mL ekleyin.</li>
		<li>TEM çubuk bağlı eşyaları kullanarak minibioreactor üzerine yükleyin.</li>
		<li>Şişe kapağı sıkın ve minibioreactor bir manyetik karıştırıcı tarih koymak. CO2 kuluçka 37 ° C'de 200 devirde minibioreactor 7-14 gün için etkinleştirin. Not: CO2 kuluçka CO2 konsantrasyonu % 5 olduğunu.</li>
	</ol>
</li>
</ol>

<ol>
	<li>Elfring, T., Boliek, C. A., Winget, M., Paulsen, C., Seikaly, H., Rieger, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+lingual+and+hypoglossal+nerve+function+and+quality+of+life+in+head+and+neck+cancer.">The relationship between lingual and hypoglossal nerve function and quality of life in head and neck cancer.</a> J. Oral Rehabil. 41, 133-140 (2014).</li><li>Patel, S. 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=Increasing+incidence+of+oral+tongue+squamous+cell+carcinoma+in+young+white+women,+Age+18+to+44+Years.">Increasing incidence of oral tongue squamous cell carcinoma in young white women, Age 18 to 44 Years.</a> J. Clin. Oncol. 29, 1488-1494 (2011).</li><li>Zhao, L., Huang, L., Yu, S., Zheng, J., Wang, H., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+tongue+tissue+as+an+in+vitro.+model+for+studying+tongue+cancer+and+tongue+regeneration.">Decellularized tongue tissue as an in vitro. model for studying tongue cancer and tongue regeneration.</a> Acta Biomaterialia. 58, 122-135 (2017).</li><li>Ng, S. L., Narayanan, K., Gao, S., Wan, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage+restricted+progenitors+for+the+repopulation+of+decellularized+heart.">Lineage restricted progenitors for the repopulation of decellularized heart.</a> Biomaterials. 32, 7571-7580 (2011).</li><li>Ott, H. 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=Perfusion-decellularized+matrix:+using+nature's+platform+to+engineer+a+bioartificial+heart.">Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart.</a> Nat. Med. 14, 213-221 (2008).</li><li>Remlinger, N. T., Wearden, P. D., Gilbert, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Procedure+for+decellularization+of+porcine+heart+by+retrograde+coronary+perfusion.">Procedure for decellularization of porcine heart by retrograde coronary perfusion.</a> J. Vis. Exp. (6), e50059 (2012).</li><li>Wainwright, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+cardiac+extracellular+matrix+from+an+intact+porcine+heart.">Preparation of cardiac extracellular matrix from an intact porcine heart.</a> Tissue Eng. Part C-ME. 16, 525-532 (2010).</li><li>Baptista, P. M., Siddiqui, M. M., Lozier, G., Rodriguez, S. R., Atala, A., Soker, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+whole+organ+decellularization+for+the+generation+of+a+vascularized+liver+organoid.">The use of whole organ decellularization for the generation of a vascularized liver organoid.</a> Hepatology. 53, 604-617 (2011).</li><li>Shupe, T., Williams, M., Brown, A., Willenberg, B., Petersen, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+the+decellularization+of+intact+rat+liver.">Method for the decellularization of intact rat liver.</a> Organogenesis. 6, 134-136 (2010).</li><li>Soto-Gutierrez, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+whole-organ+regenerative+medicine+approach+for+liver+replacement.">A whole-organ regenerative medicine approach for liver replacement.</a> Tissue Eng. Part C-ME. 17, 677-686 (2011).</li><li>Uygun, B. 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=Organ+reengineering+through+development+of+a+transplantable+recellularized+liver+graft+using+decellularized+liver+matrix.">Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix.</a> Nat. Med. 16, 814-820 (2010).</li><li>Bonvillain, R. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+nonhuman+primate+model+of+lung+regeneration:+detergent-mediated+decellularization+and+initial+in+vitro+recellularization+with+mesenchymal+stem+cells.">A nonhuman primate model of lung regeneration: detergent-mediated decellularization and initial in vitro recellularization with mesenchymal stem cells.</a> Tissue Eng. Pt A. 18, 2437-2452 (2012).</li><li>Daly, A. B., 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=Initial+binding+and+recellularization+of+decellularized+mouse+lung+scaffolds+with+bone+marrow-derived+mesenchymal+stromal+cells.">Initial binding and recellularization of decellularized mouse lung scaffolds with bone marrow-derived mesenchymal stromal cells.</a> Tissue Eng. Pt A. 18, 1-16 (2012).</li><li>Ott, H. 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=Regeneration+and+orthotopic+transplantation+of+a+bioartificial+lung.">Regeneration and orthotopic transplantation of a bioartificial lung.</a> Nat. Med. 16, 927-933 (2010).</li><li>Petersen, T. H., 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=Tissue-engineered+lungs+for+in+vivo+implantation.">Tissue-engineered lungs for in vivo implantation.</a> Science. 329, 538-541 (2010).</li><li>Price, A. P., England, K. A., Matson, A. M., Blazar, B. R., Panoskaltsis-Mortari, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+decellularized+lung+bioreactor+system+for+bioengineering+the+lung:+the+matrix+reloaded.">Development of a decellularized lung bioreactor system for bioengineering the lung: the matrix reloaded.</a> Tissue Eng. Pt A. 16, 2581-2591 (2010).</li><li>Wallis, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+assessment+of+detergent-based+protocols+for+mouse+lung+de-cellularization+and+re-cellularization.">Comparative assessment of detergent-based protocols for mouse lung de-cellularization and re-cellularization.</a> Tissue Eng. Part C-ME. 18, 420-432 (2012).</li><li>Ross, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cells+proliferate+and+differentiate+when+seeded+into+kidney+scaffolds.">Embryonic stem cells proliferate and differentiate when seeded into kidney scaffolds.</a> J. Am. Soc. Nephrol. 20, 2338-2347 (2009).</li><li>Song, J. J., Guyette, J. P., Gilpin, S., Gonzalez, G., Vacanti, J. P., Ott, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+and+experimental+orthotopic+transplantation+of+a+bioengineered+kidney.">Regeneration and experimental orthotopic transplantation of a bioengineered kidney.</a> Nat. Med. 19, 646-651 (2013).</li><li>Sullivan, D. 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=Decellularization+methods+of+porcine+kidneys+for+whole+organ+engineering+using+a+high-throughput+system.">Decellularization methods of porcine kidneys for whole organ engineering using a high-throughput system.</a> Biomaterials. 33, 7756-7764 (2012).</li><li>Soto-Gutierrez, A., Wertheim, J. A., Ott, H. C., Gilbert, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspectives+on+whole-organ+assembly:+moving+toward+transplantation+on+demand.">Perspectives on whole-organ assembly: moving toward transplantation on demand.</a> J. Clin. Invest. 122, 3817-3823 (2012).</li><li>Song, J. J., Ott, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+engineering+based+on+decellularized+matrix+scaffolds.">Organ engineering based on decellularized matrix scaffolds.</a> Trends Mol. Med. 17, 424-432 (2011).</li><li>Badylak, S. F., Taylor, D., Uygun, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-organ+tissue+engineering:+decellularization+and+recellularization+of+three-dimensional+matrix+scaffolds.">Whole-organ tissue engineering: decellularization and recellularization of three-dimensional matrix scaffolds.</a> Annu. Rev. Biomed. Eng. 13, 27-53 (2011).</li><li>Shamis, Y., 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=Organ-specific+scaffolds+for+in+vitro+expansion,+differentiation,+and+organization+of+primary+lung+cells.">Organ-specific scaffolds for in vitro expansion, differentiation, and organization of primary lung cells.</a> Tissue Eng. Part C-ME. 17, 861-870 (2011).</li><li>Nakayama, K. H., Batchelder, C. A., Lee, C. I., Tarantal, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+rhesus+monkey+kidney+as+a+three-dimensional+scaffold+for+renal+tissue+engineering.">Decellularized rhesus monkey kidney as a three-dimensional scaffold for renal tissue engineering.</a> Tissue Eng. Pt A. 16, 2207-2216 (2010).</li><li>Cortiella, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+acellular+natural+lung+matrix+on+murine+embryonic+stem+cell+differentiation+and+tissue+formation.">Influence of acellular natural lung matrix on murine embryonic stem cell differentiation and tissue formation.</a> Tissue Eng. Pt A. 16, 2565-2580 (2010).</li></ol>

Yazarlar ifşa gerek yok.

Decellularized ECM imalat için köklü bir protokol dokularda hücresel bileşenleri kaldırma sırasında yerel ECM kompozisyon tutmak gerekir neredeyse tamamen21. Perfüzyon konvektif taşıma ile hücresel malzemeleri kaldırmak için damarlara aracılığıyla gerektiren şu anda bildirilen decellularization protokolleri rağmen mekanik ajitasyon burada, geleneksel basit ve ucuz Yöntem22 bilinen kabul edilmiştir , 23 , <s...

Dil deglutition, eklem ve tatma gibi çeşitli önemli işlevleri vardır. Böylece, dil işlev bozukluğu hastaların yaşam kalitesi1üzerinde büyük etkisi vardır. Oral kavite içinde en yaygın malignite dil Skuamöz Hücre alkol veya sigara tütün2insan genellikle oluşan Karsinomu (TSCC), olduğunu.
Son yıllarda, TSCC üzerinde temel araştırma az bir gelişme sağlanmıştır. Verimli vitro araştırma eksikliği en büyük sorunlardan biri kalır modelleri. Böylece, hücre dışı matriks (ECM) olası bir çözüm olarak ortaya çıktı. ECM son derece organize matris bileşenlerden oluşan bir karmaşık ağ çerçeve olduğu bir ECM benzeri yapıda ve kompozisyon sahip İskele malzemeleri kanser araştırmaları için yetkili gerekir. Decellularized ECM mükemmel niş için hangi ECM en önemli avantajı için çıkıyor aynı kökenli vitro, hücrelerden sağlar.
ECM doku deterjanlar ve enzimler kullanarak decellularization aracılığıyla gelen kaldırılmakta olan hücresel bileşenleri ile muhafaza edilebilir. Kolajen, fibronektin ve laminin decellularized matris içinde de dahil olmak üzere çeşitli ECM bileşenleri yerel doku gibi microenvironment kültürlü hücreler için hayatta kalma, yayılması ve hücreleri3farklılaşma teşvik sağlar. Ayrıca, ECM hücresel bileşenleri yokluğu ile en az düzeye immünojenisite nakli için azaltılabilir.
Şimdiye kadar decellularized ECM imalat yöntemleri farklı doku ve organlarında, kalp4,5,6,7, karaciğer8,gibi9,10 denendi ,11, akciğer12,13,14,15,16,17ve böbrek18,19 , 20. ancak, ilgili hiçbir araştırma olmak buldu en iyi bildiğimiz kadarıyla dilinde benzer iş.
Bu çalışmada, decellularized dil hücre dışı matriks (TEM) verimli ve uygun fiyatla rezervasyon yaptırın fiziksel, kimyasal ve enzimatik tedavi bir dizi tarafından fabrikasyon. Sonra TEM TSCC vitro, özetlemek için TSCC davranış ve Kalkınma için uygun bir simülasyon gösterilen kullanıldı. TEM iyi biyouyumluluk hücreleri TEM TSCC araştırma3' te büyük potansiyeli olabilir gösterir doku özel niş Kılavuzu yeteneği vardır. Burada gösterilen iletişim kuralı Patogenez ya da TSCC klinik tedaviler üzerinde eğitim araştırmacılar için bir seçim sağlar.

<table><tbody><tr><td>C57-BL/6J mice</td><td>Sun Yat-sen University Laboratory Animal Center</td><td></td><td></td></tr><tr><td>Ethanol</td><td>Guangzhou Chemical Reagent Factory</td><td>HB15-GR-2.5L</td><td></td></tr><tr><td>Sodium chloride</td><td>Sangon Biotech</td><td>A501218</td><td></td></tr><tr><td>Potassium chloride</td><td>Sangon Biotech</td><td>A100395</td><td></td></tr><tr><td>Dibasic Sodium Phosphate</td><td>Guangzhou Chemical Reagent Factory</td><td>BE14-GR-500G</td><td></td></tr><tr><td>Potassium Phosphate Monobasic&amp;nbsp;</td><td>Sangon Biotech</td><td>A501211</td><td></td></tr><tr><td>1.5 mL EP tube</td><td>Axygen</td><td>MCT-150-A</td><td></td></tr><tr><td>Ultra-low temperature freezer&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>3.5 cm cell culture dish</td><td>Thermo Fisher Scientific</td><td>153066</td><td></td></tr><tr><td>6 cm cell culture dish</td><td>Greiner</td><td>628160</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>V900502</td><td></td></tr><tr><td>Calcium chloride</td><td>Sigma-Aldrich</td><td>746495</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich</td><td>449164</td><td></td></tr><tr><td>DNase</td><td>Sigma-Aldrich</td><td>D5025</td><td></td></tr><tr><td>Magnesium sulphate</td><td>Sangon Biotech</td><td>A601988</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>158968</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich</td><td>S5761</td><td></td></tr><tr><td>Ampicillin</td><td>Sigma-Aldrich</td><td>A9393</td><td></td></tr><tr><td>Kanamycin</td><td>Sigma-Aldrich</td><td>PHR1487</td><td></td></tr><tr><td>Surgical suture</td><td>Shanghai Jinhuan</td><td></td><td></td></tr><tr><td>250 mL wide-mouth bottle</td><td>SHUNIU</td><td>1407</td><td></td></tr><tr><td>Magnetic stirrer</td><td>AS ONE</td><td>1-4602-32</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>SHEL LAB</td><td>SCO5A</td><td></td></tr><tr><td>10 mL syringe</td><td>Hunan Pingan</td><td></td><td></td></tr><tr><td>50 mL centrifuge tube</td><td>Greiner</td><td>227270</td><td></td></tr><tr><td>Cal27 cell</td><td>Chinese Academy of Science, Shanghai Cell Bank</td><td></td><td>Tongue squamous cell carcinoma cell line</td></tr><tr><td>U2OS cell</td><td>Chinese Academy of Science, Shanghai Cell Bank</td><td></td><td>Human osteosarcoma cell line</td></tr><tr><td>DMEM/F12</td><td>Sigma-Aldrich</td><td>D0547</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Sigma-Aldrich</td><td>P5280</td><td></td></tr><tr><td>Hepes free acid</td><td>BBI</td><td>A600264</td><td></td></tr><tr><td>FBS</td><td>Hyclone</td><td>SH30084.03</td><td></td></tr><tr><td>4 &amp;deg;C fridge</td><td>Haier</td><td></td><td></td></tr><tr><td>Water purifier</td><td>ELGA</td><td></td><td></td></tr><tr><td>Hemocytometer</td><td>BLAU</td><td>717805</td><td></td></tr></tbody></table>

fabrication of tongue extracellular matrix and reconstitution of tongue squamous cell carcinoma in vitro

Dil Skuamöz Hücre Karsinomu (TSCC) vitroiçin etkili ve gerçekçi bir model oluşturmak için yöntemleri TSCC İnşaat için fonksiyonel iskele sağlayan decellularized üretmek dil hücre dışı matriks (TEM) oluşturulmuştur. TEM vitro niş hücre büyümesi, farklılaşma ve hücre geçiş sağlar. Yerel hücre dışı matriks (ECM) ve biyokimyasal besteleri decellularized matris içinde muhafaza microstructures doku özel niş hücreleri demirleme için sağlar. TEM imalatı deoksiribonükleaz (DNaz) sindirim bir ciddi organik veya inorganik Önarıtma ile eşlik tarafından gerçekleştirilebilir. Bu iletişim kuralı kullanımı kolay ve decellularization için yüksek verimlilik sağlar. TEM inşaat TSCC modeli sağlayan olumlu cytocompatibility TSCC hücrelerin çalkalanmış mı statik kültür koşullarda gösterdi. Kendini yetiştirmiş bir biyoreaktör Ayrıca hücre kültürü için kalıcı karıştırılmış koşul için kullanıldı. Yeniden oluşturulan TSCC TEM kullanarak özellikleri ve klinik TSCC histopatoloji, benzer özellikleri TSCC araştırma potansiyeli düşündüren gösterdi.

Bu iletişim kuralı TEM hazırlanması için verimli ve uygun olduğu ortaya çıkıyor. TEM anadili kurcalayarak kıyasla mükemmel decellularization gösterdi. Decellularization etkinliğini hematoksilen-(Şekil 1A-B) boyama eozin tarafından (o) doğrulandı. Sonuçları boyama o nükleer TEM (Şekil 1B) boyama tam ortadan kalkması saptandı. Ayrıca, önceki işten DNA içerik miktar DNA TE...

Watch this Scientific Journal Video about Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro at JoVE.com

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

Dil hücre dışı matriks ve dil Skuamöz Hücre Karsinomu Vitro sulandırma imalatı

Bir yöntem etkili decellularization ile dil hücre dışı matriks (TEM) hazırlanması için burada gösterilir. TEM fonksiyonel iskele dil Skuamöz Hücre Karsinomu (TSCC) manken çalkalanmış mı statik kültür koşullar altında yeniden inşası için kullanılabilir.

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce ...

fabrication-tongue-extracellular-matrix-reconstitution-tongue

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

6.2K Görüntüleme.  Sun Yat-sen University. Bir yöntem etkili decellularization ile dil hücre dışı matriks (TEM) hazırlanması için burada gösterilir. TEM fonksiyonel iskele dil Skuamöz Hücre Karsinomu (TSCC) manken çalkalanmış mı statik kültür koşullar altında yeniden inşası için kullanılabilir.

Video: Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Oral Skuamöz Hücre Karsinomu Ortotopik Fare Modeli Tümör Hücre Invasion Multi-foton Görüntüleme

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Tıp

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor microenvironment. To obtain more reliable in vitro results, several three-dimensional (3D) cell culture assays have been introduced. These assays allow cancer cells to interact with the extracellular matrix. This interaction affects cell behavior, such as proliferation and invasion, as well as cell morphology. Additionally, this interaction could induce or suppress the expression of several pro- and anti-tumorigenic molecules. Spheroid invasion assay was developed to provide a suitable 3D in vitro method to study cancer cell invasion. Currently, animal-derived matrices, such as mouse sarcoma-derived matrix (MSDM) and rat tail type I collagen, are mainly used in the spheroid invasion assays. Taking into consideration the differences between the human tumor microenvironment and animal-derived matrices, a human myoma-derived matrix (HMDM) was developed from benign uterus leiomyoma tissue. It has been shown that HMDM induces migration and invasion of carcinoma cells better than MSDM. This protocol provided a simple, reproducible, and reliable 3D human tumor-based spheroid invasion assay using the HMDM/fibrin matrix. It also includes detailed instructions on imaging and analysis. The spheroids grow in a U-shaped ultra-low attachment plate within the HMDM/fibrin matrix and invade through it. The invasion is daily imaged, measured, and analyzed using ilastik and Fiji ImageJ software. The assay platform was demonstrated using human laryngeal primary and metastatic squamous cell carcinoma cell lines. However, the protocol is suitable also for other solid cancer cell lines.

Tumor microenvironment is an essential part of cancer growth and invasion. To mimic carcinoma progression, a biologically relevant human matrix is needed. This protocol introduces an improvement for the in vitro three-dimensional spheroid invasion assay by applying a human leiomyoma-based matrix. The protocol also introduces a computer-based cell invasion analysis.

Üç boyutlu Küroid Istilası tahlil tam ınsan tümör tabanlı matris

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor ...

Kanser Araştırmaları

In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by introducing specific mutations in oncogenes and tumor suppressor genes, using clustered regulatory interspaced short palindromic repeat (CRISPR)-based genome editing technology in mice. This technology allows us to accurately mimic the genetic changes that occur in human cancers using mice. By genetically transforming murine primary cells, we can better study cancer development, progression, treatment, and diagnosis. In this study, we used Cre-inducible Cas9 mouse tongue epithelial cells to enable genome editing using adeno-associated virus (AAV) in vitro. Specifically, by altering KRAS, p53, and APC in normal tongue epithelial cells, we generated a murine head and neck cancer (HNC) cell line in vitro,which is tumorigenic in syngeneic mice. The method presented here describes in detail how to generate HNC cell lines with specific genomic alterations and explains their suitability for predicting tumor progression in syngeneic mice. We envision that this promising method will be informative and useful to study tumor biology and therapy of HNC.

Development of murine models with specific genes mutated in head and neck cancer patients is required for understanding of neoplasia. Here, we present a protocol for in vitro transformation of primary murine tongue cells using an adeno-associated virus-Cas9 system to generate murine HNC cell lines with specific genomic alterations.

Adeno-Associated Virus-Cas9 Sistemi Kullanılarak Genetiği Değiştirilmiş Bir Murine Baş ve Boyun Kanseri Hücre Hattı In Vitro Kurulması

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by ...

Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions contribute to tissue architectural changes observed during tumorigenesis remains challenging due to the lack of appropriate models. In this protocol, we describe the generation of a 3D coculture model using a LUSC primary cell culture known as TUM622. TUM622 cells were established from a LUSC patient-derived xenograft (PDX) and have the unique property to form acinar-like structures when seeded in a basement membrane matrix. We demonstrate that TUM622 acini in 3D coculture recapitulate key features of tissue architecture during LUSC progression as well as the dynamic interactions between LUSC cells and components of the tumor microenvironment (TME), including the extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs). We further adapt our principal 3D culturing protocol to demonstrate how this system could be utilized for various downstream analyses. Overall, this organoid model creates a biologically rich and adaptable platform that enables one to gain insight into the cell-intrinsic and extrinsic mechanisms that promote the disruption of epithelial architectures during carcinoma progression and will aid the search for new therapeutic targets and diagnostic markers.

An in vitro model system was developed to capture tissue architectural changes during lung squamous carcinoma (LUSC) progression in a 3-dimensional (3D) co-culture with cancer-associated fibroblasts (CAFs). This organoid system provides a unique platform to investigate the roles of diverse tumor cell-intrinsic and extrinsic changes that modulate the tumor phenotype.

Akciğer Skuamöz Karsinom Progresyonunu Modellemek Için Çok Boyutlu Coculture Sistemi

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions ...

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

Kanser Hücre Invasion Uygulama ile Tümör Stromal mikroçevresinin Doku Mühendisliği

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

Biyomühendislik

Modeling Oral-Esophageal Squamous Cell Carcinoma in 3D Organoids

Esophageal squamous cell carcinoma (ESCC) is prevalent worldwide, accounting for 90% of all esophageal cancer cases each year, and is the deadliest of all human squamous cell carcinomas. Despite recent progress in defining the molecular changes accompanying ESCC initiation and development, patient prognosis remains poor. The functional annotation of these molecular changes is the necessary next step and requires models that both capture the molecular features of ESCC and can be readily and inexpensively manipulated for functional annotation. Mice treated with the tobacco smoke mimetic 4-nitroquinoline 1-oxide (4NQO) predictably form ESCC and esophageal preneoplasia. Of note, 4NQO lesions also arise in the oral cavity, most commonly in the tongue, as well as the forestomach, which all share the stratified squamous epithelium. However, these mice cannot be simply manipulated for functional hypothesis testing, as generating isogenic mouse models is time- and resource-intensive. Herein, we overcome this limitation by generating single cell-derived three-dimensional (3D) organoids from mice treated with 4NQO to characterize murine ESCC or preneoplastic cells ex vivo. These organoids capture the salient features of ESCC and esophageal preneoplasia, can be cheaply and quickly leveraged to form isogenic models, and can be utilized for syngeneic transplantation experiments. We demonstrate how to generate 3D organoids from normal, preneoplastic, and SCC murine esophageal tissue and maintain and cryopreserve these organoids. The applications of these versatile organoids are broad and include the utilization of genetically engineered mice and further characterization by flow cytometry or immunohistochemistry, the generation of isogeneic organoid lines using CRISPR technologies, and drug screening or syngeneic transplantation. We believe that the widespread adoption of the techniques demonstrated in this protocol will accelerate progress in this field to combat the severe burden of ESCC.

This protocol describes the key steps to generate and characterize murine oral-esophageal 3D organoids that represent normal, preneoplastic, and squamous cell carcinoma lesions induced via chemical carcinogenesis.

3D Organoidlerde Oral-Özofagus Skuamöz Hücreli Karsinomun Modellenmesi

Esophageal squamous cell carcinoma (ESCC) is prevalent worldwide, accounting for 90% of all esophageal cancer cases each year, and is the deadliest of all ...

Cell Dissociation from the Tongue Epithelium and Mesenchyme/Connective Tissue of Embryonic-Day 12.5 and 8-Week-Old Mice

Cell dissociation has been an essential procedure for studies at the individual-cell level and/or at a cell-population level (e.g.,&#160;single cell RNA sequencing and primary cell culture). Yielding viable, healthy cells in large quantities is critical, and the optimal conditions to do so are tissue dependent. Cell populations in the tongue epithelium and underlying mesenchyme/connective tissue are heterogeneous and tissue structures vary in different regions and at different developmental stages. We have tested protocols for isolating cells from the mouse tongue epithelium and mesenchyme/connective tissue in the early developmental [embryonic day 12.5 (E12.5)] and young adult (8-week) stages. A clean separation between the epithelium and underlying mesenchyme/connective tissue was easy to accomplish. However, to further process and isolate cells, yielding viable healthy cells in large quantities, and careful selection of enzymatic digestion buffer, incubation time, and centrifugation speed and time are critical. Incubation of separated epithelium or underlying mesenchyme/connective tissue in 0.25% Trypsin-EDTA for 30 min at 37 &#176;C, followed by centrifugation at 200 x g for 8 min resulted in a high yield of cells at a high viability rate (&#62;90%) regardless of the mouse stages and tongue regions. Moreover, we found that both dissociated epithelial and mesenchymal/connective tissue cells from embryonic and adult tongues could survive in the cell culture-based medium for at least 3 h without a significant decrease of cell viability. The protocols will be useful for studies that require the preparation of isolated cells from mouse tongues at early developmental (E12.5) and young adult (8-week) stages requiring cell dissociation from different tissue compartments.

We have developed a generalized protocol to dissociate a large quantity of high-quality single cells from the epithelium and mesenchyme/connective tissue of embryonic and adult mouse tongues.

Embriyonik Gün 12.5 ve 8 Haftalık Farelerin Dil Epitel ve Mezenkim/Bağ Dokusundan Hücre Ayrışması

Cell dissociation has been an essential procedure for studies at the individual-cell level and/or at a cell-population level (e.g.,&#160;single cell RNA ...

Gelişim Biyolojisi

Generation and Characterization of Murine Oral Mucosal Organoid Cultures

The mucous lining covering the inside of our mouth, the oral mucosa, is a highly compartmentalized tissue and can be subdivided into the buccal mucosa, gingiva, lips, palate, and tongue. Its uppermost layer, the oral epithelium, is maintained by adult stem cells throughout life. Proliferation and differentiation of adult epithelial stem cells have been intensively studied using in vivo mouse models as well as two-dimensional (2D) feeder-cell based in vitro models. Complementary to these methods is organoid technology, where adult stem cells are embedded into an extracellular matrix (ECM)-rich hydrogel and provided with a culture medium containing a defined cocktail of growth factors. Under these conditions, adult stem cells proliferate and spontaneously form three-dimensional (3D) cell clusters, the so-called organoids. Organoid cultures were initially established from murine small intestinal epithelial stem cells. However, the method has since been adapted for other epithelial stem cell types. Here, we describe a protocol for the generation and characterization of murine oral mucosal organoid cultures. Primary epithelial cells are isolated from murine tongue tissue, embedded into an ECM hydrogel, and cultured in a medium containing: epidermal growth factor (EGF), R-spondin, and fibroblast growth factor (FGF) 10. Within 7 to 14 days of initial seeding, the resulting organoids can be passaged for further expansion and cryopreservation. We additionally present strategies for the characterization of established organoid cultures via 3D whole-mount imaging and gene-expression analysis. This protocol may serve as a tool to investigate oral epithelial stem cell behavior ex vivo in a reductionist manner.

We present a method for the generation and characterization of oral mucosal organoid cultures derived from the tongue epithelium of adult mice.

The mucous lining covering the inside of our mouth, the oral mucosa, is a highly compartmentalized tissue and can be subdivided into the buccal mucosa, ...

Biyoloji

Isolation of Circumvallate Papillae (CVP) Epithelium from Mouse Model: An Enzymatic Procedure to Separate CVP Epithelium from Mouse Tongue

Source: Shechtman, L. A. et al. <a href="https://jove.unicartagenaproxy.elogim.com/video/62300" target="_blank">Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells</a>. J. Vis. Exp. (2021)
In this video, we describe the isolation of circumvallate papillae (CVP) epithelium from a murine tongue.

Fare Modelinden Circumvallate Papilla (CVP) Epitelinin İzolasyonu: CVP Epitelini Fare Dilinden Ayırmak İçin Enzimatik Bir İşlem

Source: Shechtman, L. A. et al. Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells. J. Vis. Exp. (2021)
In this video, ...

JoVE Encyclopedia of Experiments

Kemirgen Modelleri

Yetişkin

Isolation and Culture of Murine Tongue Epithelial Cells: A Procedure to Isolate and Culture Tongue Epithelial Cells from Mouse Model

Source: Prasad, M. et al. <a href="https://jove.unicartagenaproxy.elogim.com/video/60410" target="_blank">In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line Using an Adeno-Associated Virus-Cas9 System.</a> J. Vis. Exp. (2020)
In this video, we describe the isolation of epithelial cells from a murine tongue. These cells can be further cultured to give rise to cell colonies.

Murin Dil Epitel Hücrelerinin İzolasyonu ve Kültürü: Dil Epitel Hücrelerini Fare Modelinden İzole Etme ve Kültür Prosedürü

Source: Prasad, M. et al. In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line Using an Adeno-Associated Virus-Cas9 ...