作者对国家自然科学基金 (31371390)、国家高新技术发展项目 (2014AA020702) 和广东省科技计划 (2016B030231001) 的研究资助给予了认可。

所有动物工作都是按照《动物福利法》、《机构指南》和中山大学动物保育和使用委员会批准的。
1. 制备 TEM
<ol><li>用不育的手术剪刀和镊子来执行小鼠颈椎脱位并去除舌头。</li>
	<li>浸泡在75% 乙醇3分钟的舌头, 然后把每一个舌头到1.5 毫升离心 (EP) 管与1毫升的10毫米无菌磷酸盐缓冲溶液 (PBS)。 注意: PBS 在以下所有步骤中的浓度与此步骤中的浓度相同。</li>
	<li>冷冻解冻细胞裂解: 将 EP 管中的舌头冻成-80 摄氏度, 1 小时, 然后在室温下将舌头解冻45分钟3圈。</li>
	<li>用手术针将每条舌头装入手术缝合线上, 用一小块不育的锡纸包裹缝合的末端。在不育条件下, 在含有75% 乙醇的3.5 厘米或6厘米培养皿中进行操作。 注: 每个手术缝合的适当长度约为20厘米, 每块锡纸的适当尺寸约为0.3 厘米2 (1 厘米 x 0.3 厘米)。舌头应该装在锡纸附近。</li>
	<li>三十年代用3.5 厘米或6厘米培养皿冲洗每舌3毫升的无菌 PBS. 在无菌条件下执行此操作。</li>
	<li>用超纯水冲洗舌头: 将 ampicilin 放入一个宽口瓶中, 250 毫升的无菌超纯水, 最终浓度为90µg/毫升。把舌头放进装有搅拌棒的瓶子里。拧紧瓶盖, 部分缝线留在瓶子外。在无菌条件下执行此操作。 注: 可将多达5种舌头放入同一瓶中, 考虑缠绕缝线。锡纸在瓶子的缝合端, 以防止舌头滑脱。舌头应该被放置 2 cm 高从瓶子的底部, 调整长度的缝合留在瓶子内。此注释还用于步骤1.8、1.10、1.12 和1.16。</li>
	<li>把瓶子放在磁力搅拌器上12小时。</li>
	<li>用氯化钠冲洗舌头: 将 ampicilin 添加到一个宽口瓶中, 250 毫升无菌1米氯化钠, 最终浓度为90µg/毫升。把舌头和搅拌棒放进瓶子里。拧紧瓶盖, 部分缝线留在瓶子外。在无菌条件下执行此操作。</li>
	<li>把瓶子放在磁力搅拌器上24小时。</li>
	<li>细胞裂解的海卫 X-100: 添加 ampicilin 到最后浓度的90µg/毫升成一个宽口瓶与250毫升无菌2% 海卫 X-100 在 PBS。把舌头和搅拌棒放进瓶子里。拧紧瓶盖, 部分缝线留在瓶子外。在无菌条件下执行此操作。</li>
	<li>把瓶子放在磁力搅拌器上48小时。</li>
	<li>洗舌头与 CaCl2/氯化镁2: 添加 ampicilin 到一个宽口瓶与250毫升无菌5毫米 CaCl2/氯化镁2到最后浓度90µg/毫升。把舌头和搅拌棒放进瓶子里。拧紧瓶盖, 部分缝线留在瓶子外。在无菌条件下执行此操作。</li>
	<li>把瓶子放在磁力搅拌器上24小时。</li>
	<li>DNase 消化: 添加1毫升的汉克平衡盐溶液 (HBSS) 到每个 EP 管。将 DNase 分别添加到 HBSS 中, 最终浓度为300µM. 将每一舌移入每个 EP 管, 并将部分缝合到管外。在无菌条件下执行此操作。 注: 在本步骤中, 确保保留在瓶子内的缝合部分也留在 ep 管内, 并确保在前一步骤中保持在瓶外的缝合部分也保持在本步骤的 ep 管之外。</li>
	<li>在37摄氏度的 EP 管中孵育舌头24小时。</li>
	<li>用 pbs 冲洗舌头: 将 ampicilin 加入250毫升无菌 pbs 的宽口瓶中, 最终浓度为90µg/毫升。把舌头和搅拌棒放进瓶子里。拧紧瓶盖, 部分缝线留在瓶子外。在无菌条件下执行此操作。</li>
	<li>把瓶子放在磁力搅拌器上24小时。</li>
	<li>将所制备的 TEM 在无菌 PBS 中贮存4摄氏度, 直至使用。</li>
</ol>2. 三维 (3D) TSCC 的重建
<ol><li>
静态 TSCC 模型构造
	<ol>
<li>种子 1.0 x 106单 TSCC 细胞 (Cal27) 成3.5 厘米培养皿。添加3毫升的 Dulbecco 的改良鹰的 medium/F12 (DF12), 含有10% 胎牛血清 (血清), 90 µg/毫升 ampicilin, 90 µg/毫升卡那霉素。</li>
		<li>培养的 Cal27 细胞在37°c 2 至3天。确保细胞覆盖至少60% 区域的盘子底部。</li>
		<li>将透射电镜加载到培养皿中的 Cal27 单层上。</li>
		<li>在37摄氏度的28天内, 将盘子放入 CO2孵化器中。</li>
		<li>在细胞培养过程中每天刷新培养基。在孵化器中的 CO2浓度是5%。</li>
	</ol>
</li>
	<li>
搅拌 TSCC 模型施工
	<ol>
<li>
自制搅拌 minibioreactor 的研制
		<ol>
<li>从10毫升注射器中取出柱塞。</li>
			<li>在杆的下端附近挖一个孔 (直径1厘米), 并在孔中装入搅拌杆。</li>
			<li>在塑料宽口瓶的瓶盖中央挖一个孔 (直径0.5 厘米), 并将活塞杆穿过瓶盖。</li>
			<li>切割50毫升离心管的一半, 并将其焊接在瓶盖的外侧。</li>
			<li>通过将拉杆与与鱼钩相连的钓线包裹起来, 将鱼钩连接到拉杆上。 注: 最多可连接4鱼钩杆。</li>
			<li>在使用前先将自制的复合釜压蒸。 注: 请勿蒸压塑料宽口瓶。在培养细胞时使用新的无菌塑料宽口瓶。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
动态细胞培养
	<ol>
<li>种子 1.0 x 106单 Cal27 细胞在自制的 minibioreactor。添加150毫升的 DF12 培养基, 其中含有10% 的血清, 90 µg/毫升 ampicilin, 90 µg/毫升卡那霉素。</li>
		<li>使用连接到杆上的鱼钩将 TEM 加载到 minibioreactor 上。</li>
		<li>拧紧瓶盖, 将 minibioreactor 放在磁力搅拌器上。激活 minibioreactor 在 200 rpm 在 CO2孵化器在37°c 7 到14天。 注: co2孵化器中 co2的浓度为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>

作者没有什么可透露的。

一个完善的瓣膜 ecm 制造协议应保留本机 ecm 成分, 同时去除组织中的细胞成分几乎完全21。尽管目前报告的去细胞协议需要通过血管灌流来去除蜂窝材料通过对流运输, 这里采用了机械搅拌, 称为传统的简单和廉价的方法22,23,24,25,26. 此外, 由于舌头富含 lingual...

舌有多种重要功能, 如吞咽、发音和味觉。因此, 舌功能的损害对患者的生活质量有很大的影响1。口腔内最常见的恶性肿瘤是舌鳞状细胞癌 (TSCC), 通常发生在饮酒或吸烟2的人身上。
近年来, TSCC 的基础研究取得了很小的进展。缺乏有效的体外研究模式仍然是最大的问题之一。因此, 细胞外基质 (ECM) 结果是一个潜在的解决方案。由于 ecm 是由高度组织的矩阵元件组成的复杂网络框架, 因此具有 ecm 类结构和成分的脚手架材料将胜任癌症研究。瓣膜 ecm 能够完美地为细胞从同一来源的体外提供利基, 这是 ecm 最显著的优势。
ECM 可以通过去细胞使用洗涤剂和酶将细胞成分从组织中去除。各种 ECM 组件, 包括胶原蛋白、纤连蛋白和瓣膜基质层粘连蛋白, 为培养细胞提供了一种类似于组织的微环境, 促进了细胞的存活、增殖和分化3。此外, 在 ECM 中缺乏细胞成分的情况下, 移植的免疫原性可降至最低水平。
到目前为止, 瓣膜 ECM 的制备方法已经尝试了不同的组织和器官, 如心脏4,5,6,7, 肝8,9,10 ,11, 肺12,13,14,15,16,17, 和肾脏18,19,20. 然而, 在我们最了解的情况下, 没有就舌头上的类似工作找到相关的研究。
在本研究中, 瓣膜舌外基质 (TEM) 是通过一系列物理、化学和酶处理, 高效、廉价地制备出来的。然后用透射电镜对体外TSCC 进行了重述, 对 TSCC 的行为和发育进行了适当的模拟。tem 具有良好的生物相容性以及引导细胞到组织特异性的能力, 这表明 tem 在 TSCC 研究3中可能具有很大的潜力。此处显示的协议为研究 TSCC 的发病机制或临床治疗的研究者提供了选择。

<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

为了构建一种有效的、逼真的舌鳞癌 (TSCC) 模型, 建立了瓣膜舌细胞外基质 (TEM), 为 TSCC 的构建提供了功能性支架。TEM 为细胞生长、分化和细胞迁移提供了体外小生境。本机细胞外基质 (ECM) 的显微结构和瓣膜基质中保留的生化成分为锚固细胞提供组织特异的龛位。经脱氧核糖核酸酶 (DNase) 消化, 同时具有严重的有机或无机预处理, 可实现透射电镜的制备。该协议易于操作, 并确保了去细胞的高效率。透射电镜显示, 在静态或搅拌培养条件下, TSCC 细胞具有良好的细胞相容性, 使 TSCC 模型得以构建。自制的生物反应器也用于细胞培养的持续搅拌条件。应用 TEM 重建 TSCC 显示了类似临床 TSCC 组织病理学的特点和性质, 提示 TSCC 研究的潜力。

本议定书的编写 TEM 证明是有效和适当的。与母语组织相比, 透射电镜显示出完美的去细胞。去细胞的疗效经苏木精-伊红染色 (图 1A B) 证实。he 染色结果显示透射电镜核染色完全消失 (图 1B)。此外, 从先前的工作中 dna 含量的量化表明, dna 几乎完全从 TEM3中去除。该协议在去除细胞成分?...

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

舌细胞外基质的制备与舌鳞癌体外重建

给出了一种有效去细胞的舌胞外基质 (TEM) 的制备方法。透射电镜可作为功能性支架, 用于在静态或搅拌培养条件下重建舌鳞癌 (TSCC) 模型。

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 次观看.  Sun Yat-sen University. 给出了一种有效去细胞的舌胞外基质 (TEM) 的制备方法。透射电镜可作为功能性支架, 用于在静态或搅拌培养条件下重建舌鳞癌 (TSCC) 模型。

视频: 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.

多光子成像在口腔鳞状细胞癌原位小鼠模型的肿瘤细胞的侵袭

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

科研

医学

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.

三维流状入侵检测中的全人肿瘤基基质

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

癌症研究

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.

使用腺相关病毒-Cas9系统在Vitro建立基因工程的毛因头和颈部癌细胞系

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.

多维共同培养系统模拟肺鳞癌进展

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.

肿瘤微环境基质与应用癌细胞侵袭组织工程

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

生物工程

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.

在 3D 类器官中模拟口腔-食管鳞状细胞癌

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.

从舌头上皮和梅森奇姆/胚胎日12.5和8周大的老鼠的结缔细胞分离

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 ...

发育生物学

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, ...

生物学

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.

从小鼠模型中分离环状（CVP） 上皮:从小鼠舌中分离 CVP 上皮的酶促程序

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.

小鼠舌上皮细胞的分离和培养:从小鼠模型中分离和培养舌上皮细胞的程序

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 ...

舌细胞外基质的制备与舌鳞癌体外重建

摘要

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此视频中的章节

多光子成像在口腔鳞状细胞癌原位小鼠模型的肿瘤细胞的侵袭

三维流状入侵检测中的全人肿瘤基基质

使用腺相关病毒-Cas9系统在Vitro建立基因工程的毛因头和颈部癌细胞系

多维共同培养系统模拟肺鳞癌进展

肿瘤微环境基质与应用癌细胞侵袭组织工程

在 3D 类器官中模拟口腔-食管鳞状细胞癌

从舌头上皮和梅森奇姆/胚胎日12.5和8周大的老鼠的结缔细胞分离

Generation and Characterization of Murine Oral Mucosal Organoid Cultures

从小鼠模型中分离环状（CVP）上皮:从小鼠舌中分离 CVP 上皮的酶促程序

小鼠舌上皮细胞的分离和培养:从小鼠模型中分离和培养舌上皮细胞的程序

舌细胞外基质的制备与舌鳞癌体外重建

摘要

探索更多视频

此视频中的章节

多光子成像在口腔鳞状细胞癌原位小鼠模型的肿瘤细胞的侵袭

三维流状入侵检测中的全人肿瘤基基质

使用腺相关病毒-Cas9系统在Vitro建立基因工程的毛因头和颈部癌细胞系

多维共同培养系统模拟肺鳞癌进展

肿瘤微环境基质与应用癌细胞侵袭组织工程

在 3D 类器官中模拟口腔-食管鳞状细胞癌

从舌头上皮和梅森奇姆/胚胎日12.5和8周大的老鼠的结缔细胞分离

Generation and Characterization of Murine Oral Mucosal Organoid Cultures

从小鼠模型中分离环状（CVP） 上皮:从小鼠舌中分离 CVP 上皮的酶促程序

小鼠舌上皮细胞的分离和培养:从小鼠模型中分离和培养舌上皮细胞的程序

从小鼠模型中分离环状（CVP）上皮:从小鼠舌中分离 CVP 上皮的酶促程序