Name: fabrication of tongue extracellular matrix and reconstitution of tongue squamous cell carcinoma in vitro
Uploaded: 2018-06-20T12:30:00.000+00:00
Duration: 8 min 15 s
Description: Watch this Scientific Journal Video about Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro at JoVE.com

저자는 자연 과학 재단의 중국 국가 (31371390), 상태-첨단 개발 프로젝트 (2014AA020702)의 프로그램 및 광 과학 및 기술 (2016B030231001)의 프로그램에서 연구 보조금의 지원을 인정합니다.

모든 동물 작업 동물 복지 행위, 기관 지침에 따라 수행 되었고 기관 동물 관리 및 사용 위원회, 썬 얏-센 대학교에 의해 승인.
1입니다. 준비 편
<ol><li>자 궁 경부 전위에 의해 마우스를 실행 하 고 방언 무 균 수술가 위 핀셋을 사용 하 여 제거.</li>
	<li>3 분, 75% 에탄올에 방언 담가 다음 1.5 mL으로 각 혀를 넣어 10 mM 멸 균 인산 염의 1 mL Eppendorf (EP) 튜브 버퍼링 솔루션 (PBS). 참고: 다음의 모든 단계에서 PBS의 농도이 단계에서 농도와 동일.</li>
	<li>동결 해 동 하 여 세포를 세포: 1 h-80 ° C에서 EP 튜브에 혀를 동결 하 고 다음 3 사이클에 대 일 분 동안 실내 온도에 혀를 녹여.</li>
	<li>수술 봉합 수술 바늘을 사용 하 여의 조각에 각 혀를 로드 하 고 메 마른 은박지의 작은 조각으로 봉합의 끝을 포장. 무 균 조건에서 75% 에탄올을 포함 하는 3.5 cm 또는 6 cm 문화 접시에 작업을 수행 합니다. 참고: 외과 봉합의 각 작품의 적절 한 길이 약 20cm, 그리고 각 조각의 은박지의 적절 한 크기에 대해 0.3 cm2 (1 c m × 0.3 c m). 혀는 은박지 근처 로드 합니다.</li>
	<li>30 미 수행 무 균 조건에서이 작업에 대 한 살 균 PBS의 3 mL와 함께 각 혀 3.5 cm 또는 6 cm 문화 접시에 린스.</li>
	<li>씻어 초순와 방언: 90 µ g/mL의 최종 농도에 불 임 초순의 250 mL 넓은 입 병에 ampicilin를 추가. 방언을 저 어 bar가 포함 된 병에 넣습니다. 병 밖에 남아 있는 봉합의 일부와 병 뚜껑을 조입니다. 무 균 조건에서이 작업을 수행 합니다. 참고: 5 방언까지 놓일 수 있다 봉합의 twining 고려 같은 병에. 석 박 혀 떨어져 미 끄 러에서 방지 하기 위해 병에 봉합의 끝 이다. 혀는 병 안에 남아 있는 봉합의 길이 조정 하 여 2cm 높은 병의 하단에서 배치 되어야 합니다. 이 노트 1.8, 1.10, 1.12, 및 1.16 단계 이기도합니다.</li>
	<li>12 h에 대 한 자력에 병을 넣어.</li>
	<li>NaCl와 방언 세척: 살 균 1의 250 mL 넓은 입 병에 ampicilin를 추가 M NaCl 90 µ g/mL의 최종 농도에. 병에는 방언과 볶음 바를 이동 합니다. 병 밖에 남아 있는 봉합의 일부와 병 뚜껑을 조입니다. 무 균 조건에서이 작업을 수행 합니다.</li>
	<li>24 h에 대 한 자력에 병을 넣어.</li>
	<li>트라이 톤 X-100 여 세포 세포: 살 균 2%의 250 mL와 함께 넓은 입 병에 90 µ g/mL의 최종 농도에 ampicilin를 추가 PBS에 트라이 톤 X-100. 병에는 방언과 볶음 바를 이동 합니다. 병 밖에 남아 있는 봉합의 일부와 병 뚜껑을 조입니다. 무 균 조건에서이 작업을 수행 합니다.</li>
	<li>48 h에 대 한 자력에 병을 넣어.</li>
	<li>씻어 CaCl2/MgCl2방언: 90 µ g/mL의 최종 농도에 살 균 5 m m CaCl2/MgCl2 의 250 mL 넓은 입 병에 ampicilin를 추가. 병에는 방언과 볶음 바를 이동 합니다. 병 밖에 남아 있는 봉합의 일부와 병 뚜껑을 조입니다. 무 균 조건에서이 작업을 수행 합니다.</li>
	<li>24 h에 대 한 자력에 병을 넣어.</li>
	<li>소화 DNase에 의해: 행 크의의 1 mL 균형 소금물 (HBSS) 각 EP 튜브를 추가. 각각 300의 최종 농도에 DNase HBSS로 추가 µ M.에 각 EP 튜브는 튜브 외부 봉합 부분의 각 혀 이동. 무 균 조건에서이 작업을 수행 합니다. 참고: 이전 단계에서 병 안에 남아 있는 봉합의 일부도이 단계에서 EP 튜브 안에 남아 있는지 확인 하 고 이전 단계에서 병 밖에 남아 있는 봉합의 일부도이 단계에서 EP 튜브 밖에 남아 있다 다는 것을 확인 한다.</li>
	<li>24 h에 대 한 37 ° C에서 EP 튜브에 혀를 품 어.</li>
	<li>씻어 PBS 가진 방언: 90 µ g/mL의 최종 농도에 살 균 PBS의 250 mL 넓은 입 병에 ampicilin를 추가. 병에는 방언과 볶음 바를 이동 합니다. 병 밖에 남아 있는 봉합의 일부와 병 뚜껑을 조입니다. 무 균 조건에서이 작업을 수행 합니다.</li>
	<li>24 h에 대 한 자력에 병을 넣어.</li>
	<li>사용까지 준비 편 4 ° C에서 살 균 PBS에 저장 합니다.</li>
</ol>2. TSCC의 3 차원 (3D) 재구성
<ol><li>
정적 TSCC 모델 건설
	<ol>
<li>씨 1.0 x 106 단일 TSCC 셀 (Cal27) 3.5 c m 문화 접시에. Dulbecco의의 3 mL 수정 독수리의 매체/F12 (DF12) 포함 10% 태아 둔감 한 혈 청 (FBS), 90 µ g/mL ampicilin, 및 90 µ g/mL 대 추가 합니다.</li>
		<li>2 ~ 3 일 동안 37 ° C에서 Cal27 셀 문화. 셀 60% 이상 커버 있는지 확인 접시 바닥의 지역.</li>
		<li>문화 접시에 Cal27 단층에 가장을 로드 합니다.</li>
		<li>28 일 동안 37 ° C에서 CO2 인큐베이터에 접시를 넣어.</li>
		<li>셀 문화 과정 매일 문화 매체를 새로 고칩니다. 인큐베이터에서 CO2 농도 5% 이다.</li>
	</ol>
</li>
	<li>
흔들된 TSCC 모델 건설
	<ol>
<li>
성가 냈다 minibioreactor의 준비
		<ol>
<li>10 mL 주사기에서 플런저를 꺼내.</li>
			<li>막대의 낮은 터미널 근처 구멍 (직경 1 ㎝)를 파고 및 구멍에 저 어 바 로드.</li>
			<li>플라스틱 넓은 입 병의 병 뚜껑의 중앙에 구멍 (직경 0.5 c m)를 파고 및 뚜껑을 통해 피스톤 막대를 넣어.</li>
			<li>50 mL 원심 분리기 튜브의 절반을 잘라 고 병 뚜껑의 외부 측면에 용접.</li>
			<li>fishhooks에 묶여있다는 낚시 라인 막대 여 fishhooks 막대를 연결 합니다. 참고: 4 fishhooks까지 장착할 수 막대에.</li>
			<li>압력솥 사용 하기 전에 스스로 만든된 복잡 한입니다. 참고: 플라스틱 넓은 입 병 압력솥 하지를 않습니다. 새로운 살 균 플라스틱 넓은 입 병을 사용 하 여 세포를 배양 하는 동안.</li>
		</ol>
</li>
	</ol>
</li>
	<li>
동적 세포 배양
	<ol>
<li>씨 1.0 x 106 단일 Cal27 셀 성가 minibioreactor에. 10 %FBS, 90 µ g/mL ampicilin, 및 90 µ g/mL 대 포함 된 DF12 매체의 150 mL를 추가 합니다.</li>
		<li>막대에 연결 된 fishhooks를 사용 하 여 minibioreactor에 가장을 로드 합니다.</li>
		<li>병 뚜껑을 강화 하 고는 자력에 minibioreactor를 넣어. 7 ~ 14 일 동안 37 ° C에서 CO2 배양 기에서 200 rpm에서 minibioreactor를 활성화 합니다. 참고: CO2 배양 기에서 CO2 의 농도 5%.</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>

저자는 공개 없다.

Decellularized ECM 제조에 잘 설립 프로토콜 조직에서 세포 구성 요소를 제거 하는 동안 네이티브 ECM 구성 유지 한다 거의 완전 하 게21. 현재 보고 된 decellularization 프로토콜 대류 수송에 의해 세포 물질을 제거 하는 맥 관 구조를 통해 관류를 요구 하는에 불구 하 고 기계적인 동요 채택 되었다 여기, 전통적인 간단 하 고 저렴 한 방법22 로 알려진 , <sup class="x...

혀는 deglutition, 조음, 시음 등 다양 한 중요 한 기능이 있다. 따라서, 혀 기능 장애 환자 들의 삶의 질1에 큰 영향을가지고. 구강에서 가장 일반적인 악성 종양은 혀 편평 세포 암 종 (TSCC), 일반적으로 알코올 음료 또는 담배2를 연기 하는 사람에서 생기는.
최근 몇 년 동안, 작은 진행 TSCC에 기초 연구에 달성 되었습니다. 효율적인 생체 외에서 연구의 부족 모델 가장 큰 문제 중 하나 남아 있다. 따라서, 기질 (ECM) 잠재적인 솔루션으로 밖으로 변합니다. ECM 고도로 조직된 매트릭스 구성 요소 구성 하는 복잡 한 네트워크 프레임 이기 때문에, 비 계 재료는 ECM 같은 구조와 구성 암 연구에 대 한 능력이 될 것 이다. Decellularized ECM는 같은 기원에 생체 외에서ECM의 가장 중요 한 이점을 밝혀에서 셀에 대 한 완벽 하 게 틈새를 제공할 수 있습니다.
ECM 세제와 효소를 사용 하 여 decellularization 통해 조직에서 제거 되는 세포 구성 성분으로 유지 될 수 있습니다. Decellularized 매트릭스의 콜라겐, fibronectin, 및 laminin를 포함 하 여 다양 한 ECM 구성 요소, 배양된 세포 생존, 확산, 그리고 셀3의 홍보에 대 한 기본-조직-같은 microenvironment을 제공 합니다. 또한,이 식 위한 immunogenicity ECM에 세포 구성 성분의 부재와 함께 최소한의 수준으로 줄일 수 있습니다.
지금까지, decellularized ECM에 대 한 제작 방법은 다른 조직 및 장기 심장4,5,,67년 간8,9,10 등에서 시도 되었습니다. ,11, 폐12,13,14,15,,1617및 신장18,19 , 그러나 20., 관련 연구 우리의 지식 최선 다 해 혀에서 비슷한 작품에서 찾을 수 있다.
이 연구에서는 decellularized 혀 기질 (가장) 물리, 화학, 및 효소 치료의 일련에 의해 효율적으로 그리고 싸게 조작 했다. 다음은 가장 TSCC 동작 및 개발에 대 한 적절 한 시뮬레이션을 보여주는 TSCC에서 생체 외에서정리에 사용 되었다. 가장 좋은 생체 적합성으로 가장 TSCC 연구3큰 잠재력을 가질 수 있음을 나타내는 조직-특정 틈새에 셀 가이드 수 있다. 여기에 표시 된 프로토콜 연구자 중 병 또는 TSCC의 임상 치료에 대 한 선택을 제공 합니다.

<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

혀 편평 세포 암 (TSCC) 시험관에대 한 효과적이 고 현실적인 모델을 생성 하기 위해 방법은 생산 decellularized 혀 기질 (가장) TSCC 건설 기능 건설 기계를 제공 하 게 만들어졌습니다. 가장 세포 성장, 감 별 법 및 셀 마이그레이션에 대 한 생체 외에서 틈새를 제공합니다. 원시 세포 외 기질 (ECM) 및 decellularized 매트릭스에 유지 하는 생 화 확 적인 작곡의 마이크로 구조 정박 하는 셀에 대 한 조직의 특정 틈새를 제공 합니다. 가장의 제조 deoxyribonuclease (DNase) 소화 유기 또는 무기 전처리의 심각한 동반에 의해 실현 될 수 있다. 이 프로토콜은 운영 하 게 쉬운 하 고는 decellularization에 대 한 높은 효율을 보장. 가장 TSCC 모델의 건설을 가능 하 게 TSCC 셀 정적 또는 흔들 문화 조건에 대 한 유리한 cytocompatibility를 보여주었다. 스스로 만든된 생물 세포 배양에 대 한 영구 촉발된 조건 또한 사용 되었다. 가장을 사용 하 여 복원된 TSCC 보여주었다 임상 TSCC histopathology, 닮은 속성과 특성 TSCC 연구 잠재력을 제안.

이 프로토콜의 가장 준비에 대 한 효율적이 고 적절 한 것으로 판명. 가장 완벽 한 decellularization 국어 조직 비교 했다. Decellularization의 효능 hematoxylin-오신 (그) (그림 1A-B) 얼룩에 의해 확인 되었다. 그 결과 얼룩 가장 (그림 1B)에서 핵 얼룩의 완전 한 실종을 밝혔다. 또한, 이전 작품에서 DNA 콘텐츠 정량화?...

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

혀 기질과 혀 편평 세포 암 종에서 생체 외에서 의 재구성의 제조

메서드는 효율적인 decellularization와 혀 세포 외 매트릭스 (가장)의 준비에 대 한 여기 표시 됩니다. 가장 정적 또는 흔들 문화 조건 하에서 혀 편평 세포 암 종 (TSCC) 모델의 재구성 기능 건설 기계로 사용할 수 있습니다.

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 조회수.  Sun Yat-sen University. 메서드는 효율적인 decellularization와 혀 세포 외 매트릭스 (가장)의 준비에 대 한 여기 표시 됩니다. 가장 정적 또는 흔들 문화 조건 하에서 혀 편평 세포 암 종 (TSCC) 모델의 재구성 기능 건설 기계로 사용할 수 있습니다.

비디오: 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.

구강 편평 세포 암종의 Orthotopic 마우스 모델에서 종양 세포 침략의 다중 광자 이미징

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

연구

의학

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.

3 차원 세 포스 페 로이드 침입 분석에서 완전 한 인간 종양 기반 매트릭스

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

암 연구학

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.

아데노 관련 바이러스-Cas9 시스템을 사용하여 유전자 조작 된 뮤린 머리와 목 암 세포라인의 시험관 내 설립

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.

폐 편평상피암 진행을 모델링하는 다차원 동문화 시스템

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.

암 세포의 침략에 응용 프로그램을 사용하여 종양의 기질 미시의 조직 공학

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

생체공학

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 오가노이드에서 구강-식도 편평 세포 암종 모델링

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.

배아의 날 12.5 및 8 주 된 마우스의 혀 상피 및 메센키메 / 결합 조직에서 세포 해리

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 ...

발생학

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, ...

생물학

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.

마우스 모델에서 CVP(Circumvallate Papillae) 상피의 분리: 마우스 혀에서 CVP 상피를 분리하는 효소 절차

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

Rodent Models

Adult

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.

쥐 혀 상피 세포의 분리 및 배양: 마우스 모델에서 혀 상피 세포를 분리하고 배양하는 절차

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 ...