这项工作得到了中西部大学健康科学研究促进委员会资助，授予EEH和中西部大学研究与研究局局长助学金，授予KJL。附属实验室和附属皮肤科提供了额外的支持。我们感谢Sarah Potekhen，Jamie Barto，Stefani Fawks，Cody Jording，Stacie Schimke，Heather Kissel和Ali Zaidi的技术援助。

所有对人体组织样品的实验均经Western IRB（WIRB Protocol＃20142461）批准。用于这些程序的组织收集的单一标准是SCC的活检证实的诊断。原始IRB方案中没有概述排除标准，但未使用已知血源性传染病患者的样本。在收集皮肤组织，血液和唾液之前获得知情同意书。中西部机构审查委员会批准了在西西里大学（AZ＃807）的基于临床的生物样品样品的验证工作。 皮肤肿瘤组织加工注意:这是由莫氏组织技术专家执行的。 <ol><li> 新鲜鳞状细胞癌（SCC）组织RNA和外植体培养的加工 <ol><li>组织被Mohs外科医生切除后，收获10至50毫克（≥10毫米3） </sup&gt;）组织样品从松弛期间的组织顶端侧的中心（这是操纵组织以允许其平放在载玻片上的过程）。 注意:仅当肿瘤足够大以允许在组织学评估之前移除10-50 mg大小的样品（≥10mm 2 ），这是Mohs手术的一部分，才有可能。如果肿瘤不够大以至少要移除2mm 3 ，则不要进行RNA的处理（跳过步骤5）。 </li><li>使用镊子将活组织转移到含有培养基（DMEM:Ham&#39;s F-12,25mM HEPES，100IU / mL的青霉素和100μg/ mL链霉素的1:1混合物）的标记的1.5mL管中，并确保组织完全浸没在培养基中。将此管存放在4°C，然后再进行加工。 </li><li>使用组织进行RNA分离和表达分析（见步骤5）和/或建立在24小时内进行外植体培养（见步骤6）。 </li><li>将从患者中取出的肿瘤样品的剩余部分（步骤1.1.1）放置在低温恒温器组织支架（或"卡盘"）上。将组织嵌入最佳切割温度（OCT）化合物中，冷冻保持器内的组织，以允许完成莫氏组织学和制备用于实验分析的另外的组织学载玻片。 </li></ol></li><li> 组织学幻灯片的制备 <ol><li>使用低温恒温器切割7μm厚的组织切片，并为每个肿瘤样品创建两个（或更多）个载玻片。确保每个幻灯片最多包含完整肿瘤的三个部分。 注意:两个幻灯片中的一个将被Mohs组织技术学家用H＆E染色，另外的幻灯片可以用IF，IHC或FISH分析进行处理。 </li><li>将载玻片放入丙酮中1-2s以固定组织。将这些幻灯片放在一边干燥。不要H＆E染色或盖玻片以用于将来的免疫荧光（IF），免疫组织化学（IHC）或荧光原位杂交（FISH）分析，这取决于实验方案。 </li></ol></li><li> 后Mohs组织的处理 <ol><li>一旦Mohs处理完成（步骤1.1-1.2），在低温恒温室中工作，并从低温包埋介质中撬起样品并将组织置于标记的低温管中。 </li><li>在组织解冻之前，将标记的小瓶置于液氮中。如果将样品用于蛋白质和/或DNA分析，则可将其转移到液氮或-80℃冰箱中，以便将来储存以用于将来的蛋白质表达和DNA突变分析（参见步骤4-5） 。 注意:如果从Mohs后期样品中需要完整的RNA，则必须在将样品从冷冻包埋介质中取出时保持冷冻。如果不行使极端的照顾此步骤后Mohs样品应保留用于蛋白质和/或DNA分析。 </li></ol></li><li> 新鲜ANT的加工 <ol><li>在Mohs关闭时，从Mohs外科医生那里获得ANT。 </li><li>从正常组织的顶端取出从SCC样品（见上文）收获的等量或更大量的ANT，并将其转移到含有培养基（1:1混合DMEM:Ham&#39;s F- 12，补充有25mM HEPES，100IU / mL青霉素和100μg/ mL链霉素）。 </li><li>将组织完全浸入同一培养基中并在4℃下储存，直到后续处理（见步骤4-6）。 </li></ol></li></ol>血液，唾液和颊拭子加工<ol><li>通过静脉穿刺在EDTA收集管中获得约10 mL的血液。 </li><li> 将血液从收集管转移到无菌的15或50 mL离心管中。将大约200μL去除在1.5 mL低温管中，避免气泡;将这种全血样品储存在-80°C，如有必要可用于将来的基因分型和组织匹配。 <ol><li>将剩余血液以1,000xg旋转30分钟，并按照标准程序收集血浆级分。 </li><li>在1.5 mL低温管中将等离子体等分为1 mL增量，并储存于-80°C，以备将来进行液体活检分析。 </li></ol></li><li>使用无菌拭子获得颊拭子样品，并将拭子在室温下储存在无菌管中。 </li><li>如果需要，获得唾液样品，并在室温下储存在无菌管中。 注意:如有必要，这些样品可用于将来的基因分型和组织匹配。 </li></ol> 3.记录样品<ol><li>将患者样本清单中的信息转移到生物参考数据库。包括患者信息和诊断nosis从病人图。 </li><li>使用每个样品的条形码打印标签，并将数据记录在数据库中，以将每个收集的样本与患者数据相链接。 </li><li>在将样品转移到研究实验室之前，记录未确定样品在生物数据库中的分布情况。 </li></ol>蛋白质分离和蛋白质印迹分析<ol><li>将每个组织样品（SCC和/或ANT）放入含有每毫升组织含有蛋白酶抑制剂的500μL细胞裂解缓冲液的小瓶中以裂解组织。 注意:我们使用由50mM Tris pH 8.0,150mM NaCl，0.5％脱氧胆酸钠，1.0％NP-40和0.1％SDS组成的放射免疫沉淀测定（RIPA）缓冲液。可以使用其它细胞裂解缓冲液。 </li><li>使用组织匀浆器在4℃以20秒间隔使组织匀化5分钟。将样品在21,000 xg和4℃下旋转20分钟，并将含有蛋白质的上清液收集到新鲜的tu中是。使用相同的条件重新旋转上清液，并将最终的上清液收集到新鲜管中。 </li><li>使用4-20％SDS-聚丙烯酰胺梯度凝胶从每个样品中电泳30-50μg蛋白质。如果需要，用考马斯蓝染色凝胶以显现蛋白条带。 </li><li>如果需要蛋白质印迹分析表达的蛋白质，按照标准方案将蛋白质从凝胶转移到聚偏二氟乙烯（PVDF）膜。转移后，如果需要，Ponceau染色PVDF膜以显现蛋白条带。 </li><li>阻止PVDF膜和探针以按照标准方案检测目标蛋白质。按照制造商推荐的蛋白质印迹分析的浓度，使用对所关注的蛋白质特异性的一级抗体以及对于一级抗体特异的相应的二级抗体。 </li><li>使用化学发光底物暴露探测膜，并使用图像对膜进行成像ng系统。 </li></ol> RNA分离和定量聚合酶链反应（qPCR） <ol><li>立即将新鲜的组织样本（SCC和/或ANT）放入含有RNA稳定溶液的1.5mL管中，并将Mohs组织样品放入含有冷冻组织转换溶液的1.5mL管中，按照制造商的建议。确保组织完全浸没。将这些样品储存在-80°C直到使用。 </li><li>从-80°C取出RNA样品，并在冰上解冻。将组织转移到每50-100mg组织中含有1mL RNA提取缓冲液（苯酚/硫氰酸胍）的1.5mL管中。 </li><li>使用组织匀浆器裂开组织。进行六个循环的均质化，然后进行一段时间的样品冷却;每个循环由4℃均匀化和40s冷却期20s组成。均化后，在室温下孵育样品5分钟。 </li><li>加0.2mL氯仿/ 1mL RNA提取缓冲液，用手剧烈摇动管15秒。在室温下孵育2-3分钟。 </li><li>在12,000 xg和4℃下旋转样品15分钟。取出样品的水相并将其放入新的1.5 mL管中。 </li><li>每1mL步骤5.2中使用的RNA提取缓冲液加入0.5mL 100％异丙醇至水相，并在室温下孵育10分钟。 注意:如果RNA产量预计为低（ 即起始组织体积小于10毫克），样品可能会在-80℃下沉淀过夜。另外，在异丙醇沉淀步骤期间，可以加入每500μLRNA提取缓冲液中5μg无RNA酶的糖原，以提高RNA产量。 </li><li>在4℃下以12,000xg旋转样品10分钟。取出上清液，用1 mL 75％乙醇洗涤沉淀。 </li><li>短暂旋转样品，然后以7,500 xg和4°旋转C 5分钟弃去洗涤物并将其干燥5-10分钟。 </li><li> 将RNA沉淀重悬于20μL无RNase的水中。 <ol><li>如果需要，按照制造商的建议，使用RNA清洁试剂盒纯化每个RNA样品。 注意:如果执行此步骤，小RNA将从样品中丢失。 </li></ol></li><li>使用1.2％甲醛-MPS琼脂糖凝胶电泳1μg样品以分析RNA的质量。 注意:作为替代方案，使用生物分析仪分析样品以获得RIN编号13 。 </li><li>使用反转录方案将RNA逆转录为cDNA，以便对感兴趣的靶mRNA（或miRNA）进行qPCR分析。 </li></ol> 6.组织外植体培养<ol><li>在将组织浸入70％乙醇中之前，将培养前的组织进行灭菌。 </li><li>将组织放在载玻片或碟上，加入足够的培养基（DMEM:Ham＆＃39; s F-12，补充有10％FBS，25mM HEPES，100IU / mL青霉素和100μg/ mL链霉素）以覆盖组织（以防止组织干燥）。使用解剖刀刀片小心地将组织切成碎片（&lt;1 mm 3 ）。 </li><li>将组织碎片转移到35mm组织培养皿中，并在每个片段的顶部加入约20μL胎牛血清（FBS）。 </li><li>将培养皿放在培养罩中20-30分钟，或直到几乎干燥。 </li><li>向每个培养皿中加入1mL培养基（1:1混合DMEM:Ham&#39;s F-12，补充有10％FBS，25mM HEPES，100IU / mL青霉素和100μg/ mL链霉素），并在37℃下孵育C在5％CO 2中 。如果需要，进行亚文化或亚克隆培养。 </li></ol>

<ol>
	<li>Baker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biorepositories:+Building+better+biobanks.">Biorepositories: Building better biobanks.</a> Nature. 486, 141-146 (2012).</li><li>Ambrosone, C. B., Nesline, M. K., Davis, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+a+cancer+center+data+bank+and+biorepository+for+multidisciplinary+research.">Establishing a cancer center data bank and biorepository for multidisciplinary research.</a> Cancer Epidemiol Biomarkers Prev. 15, 1575-1577 (2006).</li><li>Baird, P. M., Gunter, E. W., Vaught, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+a+biobank.">Building a biobank.</a> Biopreserv Biobank. 14, 87-88 (2016).</li><li>Caixeiro, N. J., Lai, K., Lee, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quality+assessment+and+preservation+of+RNA+from+biobank+tissue+specimens:+a+systematic+review.">Quality assessment and preservation of RNA from biobank tissue specimens: a systematic review.</a> J Clin Pathol. 69, 260-265 (2016).</li><li>Campbell, L. D., 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=Development+of+the+ISBER+best+practices+for+repositories:+Collection,+storage,+retrieval+and+distribution+of+biological+materials+for+research.">Development of the ISBER best practices for repositories: Collection, storage, retrieval and distribution of biological materials for research.</a> Biopreserv Biobank. 10, 232-233 (2012).</li><li>Neumeister, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+to+assess+tissue+quality.">Tools to assess tissue quality.</a> Clin Biochem. 47, 280-287 (2014).</li><li>Vaught, 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=An+NCI+perspective+on+creating+sustainable+biospecimen+resources.">An NCI perspective on creating sustainable biospecimen resources.</a> J Natl Cancer Inst Monogr. 2011, 1-7 (2011).</li><li>Fu, T., Aasi, S. Z., Hollmig, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+high-risk+squamous+cell+carcinoma+of+the+skin.">Management of high-risk squamous cell carcinoma of the skin.</a> Curr Treat Options Oncol. 17, (2016).</li><li>Rasmussen, C., Thomas-Virnig, C., Allen-Hoffmann, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classical+human+epidermal+keratinocyte+cell+culture.">Classical human epidermal keratinocyte cell culture.</a> Methods Mol Biol. 945, 161-175 (2013).</li><li>Purdie, K. J., Pourreyron, C., South, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+cell+culture:+Methods+and+protocols.">Cancer cell culture: Methods and protocols.</a> , 151-159 (2011).</li><li>Berglund, S. R., Schwietert, C. W., Jones, A. A., Stern, R. L., Lehman, J., Goldberg, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+methodology+for+sequential+extraction+of+RNA+and+protein+from+small+human+skin+biopsies.">Optimized methodology for sequential extraction of RNA and protein from small human skin biopsies.</a> J Invest Dermatol. 127, 349-353 (2007).</li><li>Schroeder, 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=The+RIN:+an+RNA+integrity+number+for+assigning+integrity+values+to+RNA+measurements.">The RIN: an RNA integrity number for assigning integrity values to RNA measurements.</a> BMC Mol Biol. 7, 1-14 (2006).</li><li>Cooper, H. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+glycosylation+of+MUC1+in+epidermolysis+bullosa-associated+and+sporadic+cutaneous+squamous+cell+carcinomas.">Expression and glycosylation of MUC1 in epidermolysis bullosa-associated and sporadic cutaneous squamous cell carcinomas.</a> Br J Dermatol. 151, 540-545 (2004).</li><li>Riihila, P. 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=Complement+factor+H:+a+biomarker+for+progression+of+cutaneous+squamous+cell+carcinoma.">Complement factor H: a biomarker for progression of cutaneous squamous cell carcinoma.</a> J Invest Dermatol. 134, 498-506 (2014).</li><li>Ha Lan, T. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+p40+isoform+of+p63+has+high+specificity+for+cutaneous+sarcomatoid+squamous+cell+carcinoma.">Expression of the p40 isoform of p63 has high specificity for cutaneous sarcomatoid squamous cell carcinoma.</a> J Cutan Pathol. 41, 831-838 (2014).</li><li>Alomari, A. K., Glusac, E. J., McNiff, 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=p40+is+a+more+specific+marker+than+p63+for+cutaneous+poorly+differentiated+squamous+cell+carcinoma.">p40 is a more specific marker than p63 for cutaneous poorly differentiated squamous cell carcinoma.</a> J Cutan Pathol. 41, 839-845 (2014).</li><li>Qiao, B., Johnson, N. W., Gao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-mesenchymal+transition+in+oral+squamous+cell+carcinoma+triggered+by+transforming+growth+factor-beta1+is+Snail+family-dependent+and+correlates+with+matrix+metalloproteinase-2+and+-9+expressions.">Epithelial-mesenchymal transition in oral squamous cell carcinoma triggered by transforming growth factor-beta1 is Snail family-dependent and correlates with matrix metalloproteinase-2 and -9 expressions.</a> Int J Oncol. 37, 663-668 (2010).</li><li>Vaught, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developments+in+biospecimen+research.">Developments in biospecimen research.</a> Br Med Bull. 114, 29-38 (2015).</li></ol>

作者没有什么可以披露的。

据笔者了解，该协议是首例以成本效益和速度快的方式重点关注皮肤组织样本的临床采购。经历莫氏显微外科手术的患者通常在特定的时间段内安排，收集限于这些时期。样本收集涉及病人护理中涉及的医疗助理，处理肿瘤样本的莫氏组织学技术人员以及处理样品进入和血液处理的指定实验室工作人员的努力。 收集队的三名成员（ 即医疗助理，莫斯组织技术员和实验?...

癌症和生物标志物研究依赖于提供优质的人体组织样本，有限的供应阻碍了研究1,2 。许多皮肤病学研究受到供应不足，质量变化以及与使用人体组织有关的成本的限制。建立大型专用生物库的成本估计约为200万美元3 ，这些成本将人体组织的使用放在许多研究人员的触角之外。此外，生成和存储研究样本的过程有可能影响临床操作，并延迟患者护理，如果不慎重执行。已经建立了一个具有成本效益，基于诊所的生物反应器，其重点是按照推荐的最佳实践和样品验证4,5,6 。 <pclass ="jove_content"&gt;该协议已经在皮肤科诊所中开发，该诊所执行大量的Mohs显微手术以去除鳞状细胞癌（SCC），基底细胞癌（BCC）和黑素瘤皮肤癌。志愿者捐助者可以从这个病人群中招募。重要的是在收集部位建立生物反应器，以迅速从同意的患者中获取组织和血液，而不会延迟治疗。收集相同诊所的样品可最大限度地减少采集技术的变化，并最大限度地减少样品质量的变化，这对于下游应用7,8可能是有问题的。 莫氏显微手术技术的目的是确保所有的癌组织被去除，同时保留尽可能多的健康组织。该过程涉及逐渐去除肿瘤组织的薄层。每个连续的层是他的通过皮肤科医师进行病理学检查（在对肿瘤组织进行冷冻切片并进行H＆E染色后），以确定是否所有的癌组织都被去除。当患者留在办公室时，执行随后的组织层的切除和检查。该技术被认为是SCC 9的最佳治疗方案。此时，伤口闭合，为了改善愈合和美容外观，常常切除相邻的正常组织（ANT）。因此，这种去除肿瘤的外科手术理想地适用于收集组织学表征的组织用于将来的研究。 用于获得肿瘤组织，相邻正常组织，唾液和血液样本的采购程序被设计为对正常人员职责的影响最小（ 图1 ）。医疗助理在准备患者手术时进行抽血。在完成莫氏程序后，<html>哦，组织技术人员准备标本的附加组织学载玻片并将组织转移到生物支架。与建立生物反应器相关的成本包括购买冷冻保存冷冻机，创建适度的临床实验室空间以及开发库存跟踪计划。 <img alt="图1" src="/files/ftp_upload/55583/55583fig1.jpg" /> 图1:样本收集和负责人员的顺序。病人登记并取得病人同意后，医疗助理将收集颊拭子并进行抽血。然后皮肤科医师和医疗助理人员切除肿瘤并关闭伤口，在此期间分别收集SCC和ANT标本。专门的实验室技术人员处理血液和切片SCC和ANT标本，用于组织培养，保存和进入生物支架。ftp_upload / 55583 / 55583fig1large.jpg"target ="_ blank"&gt;请点击此处查看此图的较大版本。 收集的样品的多样性可实现多种实验方法（ 图2 ）。从患者收集的样品是颊拭子（如果需要，也可以收集唾液），全血和切除的组织。颊拭子和来自全血的样品在不加工的情况下被保存用于基因分型和组织匹配。将全血分离成白细胞（WBC）和血浆级分用于未来分析。在Mohs加工之后，将冷冻的肿瘤直接置于液氮中并转移至-80℃的冷冻器中。使用先前技术10,11的修饰培养新鲜可行的肿瘤组织和ANT样品，然后冷冻保存。在收集期间，每个样本类型的数量在进入之前记录在电子表格上<html>库存跟踪程序便于准确处理（ 表1 ）。 <img alt="图2" src="/files/ftp_upload/55583/55583fig2.jpg" /> 图2:基于临床的生物样品采集和处理的概述。从患者收集颊拭子和血液样品，并存储用于下游基因分型和组织匹配。全血进一步处理白细胞（WBC）分离和CTC分析，以及血浆采集和液体活检分析。在Mohs手术期间切除的组织进行组织学处理以用于诊断目的，之后组织学载玻片可用于实验用于进一步的免疫组织化学分析。假设切除的组织样本足够大，一部分新鲜组织被去除并切片以进行蛋白质和RNA分离，并建立培养的细胞系。55583 / 55583fig1large.jpg"target ="_ blank"&gt;请点击此处查看此图的较大版本。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>收藏日期: </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td>病人1 </td><td>病人2 </td><td>病人3 </td><td>病人4 </td><td>病人5 </td></tr><tr><td>姓名和出生日期</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>眼睛的颜色</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>样品类型</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>样品位置</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>唾液</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> w ^洞血</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>等离子体</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>肿瘤生存</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>组织正常生存</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>肿瘤莫氏液氮</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>组织正常液氮</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>幻灯片</td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table> 表1:记录样品收集的清单。收集的每个样本跟踪和记录的数据包括患者姓名，出生日期和眼睛颜色（皮肤分类）以及位置取样。唾液样本的数量，采血量以及收集的活的和保存的组织标本的数量也被记录作为后续用途分配的参考。 <a href="http://ecsource.jove.com/files/ftp_upload/55583/Table1.xlsx">请点击这里下载此文件。</a> 为了验证样品收集程序，已经在下游应用中测试了每种样品类型。使用先前技术的修改12 ，肿瘤和ANT已经成功地用于蛋白质和RNA分离，并且可以潜在地用于DNA分离。已经通过显微镜评估从组织切片建立的活的外植体，而存储的组织学载玻片已被用于免疫组织化学和免疫荧光。 通过遵循这里描述的方案，可以将该模型扩展到其他皮肤科诊所，其他肿瘤类型（如黑素瘤），以及其他外科专业和实践，为人类癌症的多方面研究提供人体组织样本。对于其他实践，可能需要对该协议进行轻微的修改，但原则上该协议适用于常规地丢弃患者治疗过程中收集的患者样本的任何外科手术。

<table><tbody><tr><td>BD Vacutainer Plastic Blood Collection Tubes with K&lt;sub&gt;3&lt;/sub&gt;EDTA</td><td>ThermoFisher Scientific</td><td>02-685-2B</td><td></td></tr><tr><td>Electron Microscopy Sciences Double Edge Blades</td><td>ThermoFisher Scientific</td><td>50-949-411</td><td></td></tr><tr><td>Curved Medium Point General Purpose Forceps</td><td>ThermoFisher Scientific</td><td>16-100-110</td><td></td></tr><tr><td>Premium Microcentrifuge Tubes</td><td>ThermoFisher Scientific</td><td>05-408-138</td><td></td></tr><tr><td>Lonza Walkersville KGM Keratinocyte Medium</td><td>ThermoFisher Scientific</td><td>NC9791321</td><td></td></tr><tr><td>Electron Microscopy Sciences Tissue TEK OCT Compound</td><td>ThermoFisher Scientific</td><td>50-363-579</td><td></td></tr><tr><td>Leica CM1950 Cryostat</td><td>Leica Biosystems</td><td>14047743909</td><td></td></tr><tr><td>Frosted Microscope Slides</td><td>ThermoFisher Scientific</td><td>12-550-343</td><td></td></tr><tr><td>Shandon Rapid-Chrome H&amp;amp;E Frozen Section Staining Kit</td><td>ThermoFisher Scientific</td><td>99-900-01</td><td></td></tr><tr><td>Nalgene Long-Term Storage Cryogenic Tubes</td><td>ThermoFisher Scientific</td><td>03-337-7X</td><td></td></tr><tr><td>Falcon 15 mL Conical Centrifuge Tubes</td><td>ThermoFisher Scientific</td><td>14-959-53A</td><td></td></tr><tr><td>Boca Scientific BuccalT-Swab&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>NC9679349</td><td></td></tr><tr><td>Cell Signaling Technology 10x RIPA Buffer</td><td>ThermoFisher Scientific</td><td>50-195-822</td><td></td></tr><tr><td>Halt Protease and Phosphatase Inhibitor Cocktail</td><td>ThermoFisher Scientific</td><td>PI78443</td><td></td></tr><tr><td>Eppendorf 5424R Microcentrifuge</td><td>ThermoFisher Scientific</td><td>05-401-203</td><td></td></tr><tr><td>Pellet Morter Cordless Homogenizer</td><td>ThermoFisher Scientific</td><td>12-141-3</td><td></td></tr><tr><td>RNAse Free Pellet Pestle</td><td>ThermoFisher Scientific</td><td>121-141-364</td><td></td></tr><tr><td>Forma SteriCycle CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>ThermoFisher Scientific</td><td>13-998-089</td><td></td></tr><tr><td>HyClone Fetal Bovine Serum</td><td>ThermoFisher Scientific</td><td>SH30071.02</td><td></td></tr><tr><td>Gibco Advanced DMEM/F-12</td><td>ThermoFisher Scientific</td><td>12-634-028</td><td></td></tr><tr><td>Gibco Penicillin-Streptomycin</td><td>ThermoFisher Scientific</td><td>15140148</td><td></td></tr><tr><td>Gibco 1 M Hepes</td><td>ThermoFisher Scientific</td><td>15-630-130</td><td></td></tr><tr><td>Nunc Cell Culture 35 mm with Vent</td><td>ThermoFisher Scientific</td><td>1256591</td><td></td></tr><tr><td>Anti-CFH monoclonal antibody clone OX-24</td><td>Abnova</td><td>MAB12583</td><td></td></tr><tr><td>Anti-p40 (p63 delta) antibody</td><td>Abnova</td><td>ABX-144A</td><td></td></tr><tr><td>Anti MUC-1 antibody</td><td>Santa Cruz Biotech</td><td>sc-7313</td><td></td></tr><tr><td>Anti-Snail + Slug (phospho S246)</td><td>Abcam</td><td>Ab63568</td><td></td></tr><tr><td>Goat anti-rabbit IgG, Alexa Fluor 488</td><td>Invitrogen</td><td>A-11034</td><td></td></tr><tr><td>Alexa Fluor 568 Phallodin</td><td>Molecular Probes&amp;nbsp;</td><td>A12380</td><td></td></tr><tr><td>ProLong Gold Antifade Mountant with DAPI</td><td>Molecular Probes&amp;nbsp;</td><td>P36941</td><td></td></tr><tr><td>Zeiss Axio Imager Z1 Microscope with Axiocam camera</td><td>Zeiss</td><td>4300009901</td><td></td></tr><tr><td>Olympus IX51 phase contrast with DP72 camera</td><td>Olympus</td><td>IX511F3</td><td></td></tr></tbody></table>

establishment of a clinic-based biorepository

在过去几年中，皮肤癌（ 例如鳞状细胞癌，基底细胞癌和黑素瘤）的发病率一直在增加。预计对于生物医学研究研究的皮肤肿瘤样本将会有平行的需求。然而，由于建立生物制剂的成本和缺乏可用于获得不干扰临床操作的临床样品的方案，组织的可用性是有限的。建立了一项协议，收集和处理在莫氏手术之日对常规临床手术影响最小的皮肤肿瘤和相关血液和唾液样品。 Mohs组织学技术人员从经历第一层莫氏手术的患者收集并加工肿瘤样本，用于活检证实的皮肤恶性肿瘤。在手术闭合时收集相邻的正常组织。可能收集的其他样品是全血和颊拭子。通过使用通常丢弃的组织样本，生成生物反应器，其通过基于临床和实验室设置提供了几个关键优点。这些包括广泛收集的样品;获取未确定的患者记录，包括病理报告;而对于典型的捐助者，在后续访问期间可以获得额外的样本。

肿瘤和ANT已经成功地用于蛋白质分离，并通过Western印迹进行测试。 如图3所示 ，可以在收集并存储在我们基于临床的生物反应器中的患者样品中检测到皮肤鳞状细胞癌标志物（Muc1 14 ，FHL- 115和p40 16,17 ）。 <img alt="图3" src="/files/ftp_upload/55583/55583fig3.jpg" /> <st...

Watch this Scientific Journal Video about Establishment of a Clinic-based Biorepository at JoVE.com

Establishment of a Clinic-based Biorepository

经Mohs显微手术后，皮肤肿瘤经常被丢弃。这里描述了一种方案，使临床支持人员有效地处理和存储下游实验室应用的皮肤肿瘤（ 如鳞状细胞癌，基底细胞癌和黑色素瘤）样品，而不会干扰临床操作。

建立以临床为基础的生物制剂

The incidence of skin cancer (e.g., squamous cell carcinoma, basal cell carcinoma, and melanoma) has been increasing over the past several years. It is ...

establishment-of-a-clinic-based-biorepository

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

6.4K 次观看.  Midwestern University. 经Mohs显微手术后，皮肤肿瘤经常被丢弃。这里描述了一种方案，使临床支持人员有效地处理和存储下游实验室应用的皮肤肿瘤（ 如鳞状细胞癌，基底细胞癌和黑色素瘤）样品，而不会干扰临床操作。 

视频: Establishment of a Clinic-based Biorepository

Generation of Comprehensive Thoracic Oncology Database - Tool for Translational Research

The Thoracic Oncology Program Database Project was created to serve as a comprehensive, verified, and accessible repository for well-annotated cancer specimens and clinical data to be available to researchers within the Thoracic Oncology Research Program. This database also captures a large volume of genomic and proteomic data obtained from various tumor tissue studies. A team of clinical and basic science researchers, a biostatistician, and a bioinformatics expert was convened to design the database. Variables of interest were clearly defined and their descriptions were written within a standard operating manual to ensure consistency of data annotation. Using a protocol for prospective tissue banking and another protocol for retrospective banking, tumor and normal tissue samples from patients consented to these protocols were collected. Clinical information such as demographics, cancer characterization, and treatment plans for these patients were abstracted and entered into an Access database. Proteomic and genomic data have been included in the database and have been linked to clinical information for patients described within the database. The data from each table were linked using the relationships function in Microsoft Access to allow the database manager to connect clinical and laboratory information during a query. The queried data can then be exported for statistical analysis and hypothesis generation.

A thoracic oncology database was developed to serve as a comprehensive repository for clinical and laboratory data for the purposes of translational research. The database will serve translational cancer researchers within the Thoracic Oncology Research Program. This database is adaptable to other cancer models, as well as other human diseases.

综合胸部肿瘤生成数据库 - 翻译研究的工具

The Thoracic Oncology Program Database Project was created to serve as a comprehensive, verified, and accessible repository for well-annotated cancer ...

科研

医学

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

在免疫缺陷小鼠的建立和传播人类视网膜母细胞瘤肿瘤

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

Creation and Maintenance of a Living Biobank - How We Do It

In light of the growing knowledge about the inter-individual properties and heterogeneity of cancers, the emerging field of personalized medicine requires a platform for preclinical research. Over recent years, we have established a biobank of colorectal and pancreatic cancers comprising of primary tumor tissue, normal tissue, sera, isolated peripheral blood lymphocytes (PBL), patient-derived xenografts (PDX), as well as primary and secondary cancer cell lines. Since original tumor tissue is limited and the establishment rate of primary cancer cell lines is still relatively low, PDX allow not only the preservation and extension of the biobank but also the generation of secondary cancer cell lines. Moreover, PDX-models have been proven to be the ideal in vivo model for preclinical drug testing. However, biobanking requires careful preparation, strict guidelines and a well attuned infrastructure. Colectomy, duodenopancreatectomy or resected metastases specimens are collected immediately after resection and transferred to the pathology department. Respecting priority of an unbiased histopathological report, at the discretion of the attending pathologist who carries out the dissections, small tumor pieces and non-tumor tissue are harvested. 
Necrotic parts are discarded and the remaining tumor tissue is cut into small, identical cubes and cryopreserved for later use. Additionally, a small portion of the tumor is minced and strained for primary cancer cell culture. Additionally, blood samples drawn from the patient pre- and postoperatively, are processed to obtain serum and PBLs. For PDX engraftment, the cryopreserved specimens are defrosted and implanted subcutaneously into the flanks of immunodeficient mice. The resulting PDX closely recapitulate the histology of the "donor" tumors and can be either used for subsequent xenografting or cryopreserved for later use. In the following work, we describe the individual steps of creation, maintenance and administration of a large biobank of colorectal and pancreatic cancer. Moreover, we highlight the crucial details and caveats associated with biobanking.

In the following work, we describe the consecutive steps necessary for the establishment of a large biobank of colorectal and pancreatic cancer.

活生物库的创建和维护 - 我们如何做到这一点

In light of the growing knowledge about the inter-individual properties and heterogeneity of cancers, the emerging field of personalized medicine requires ...

癌症研究

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

微创建立小鼠原位膀胱移植瘤的

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

A Standardized Liquid Biopsy Preanalytical Protocol for Downstream Circulating-Free DNA Applications

The term liquid biopsy (LB) refers to molecules such as proteins, DNA, RNA, cells, or extracellular vesicles in blood and other bodily fluids that originate from the primary and/or metastatic tumor. LB has emerged as a mainstay in translational research and has started to become part of clinical oncology practice, providing a minimally invasive alternative to solid biopsy. The LB allows real-time monitoring of a tumor via a minimally invasive sample extraction, such as blood. The applications include early cancer detection, patient follow-up for the detection of disease progression, assessment of minimal residual disease, and potential identification of molecular progression and mechanism of resistance. In order to achieve a reliable analysis of these samples that can be reported in the clinic, the preanalytical procedures should be carefully considered and strictly followed. Sample collection, quality, and storage are crucial steps that determine their usefulness in downstream applications. Here, we present standardized protocols from our liquid biopsy working module for collecting, processing, and storing plasma and serum samples for downstream liquid biopsy analysis based on circulating-free DNA. The protocols presented here require standard equipment and are sufficiently flexible to be applied in most laboratories focused on biological procedures.

The liquid biopsy has revolutionized our approach to oncology translational studies, with sample collection, quality, and storage being crucial steps for its successful clinical application. Here we describe a standardized and validated protocol for downstream circulating-free DNA applications that can be applied in most translational research laboratories.

用于下游无循环DNA应用的标准化液体活检前分析方案

The term liquid biopsy (LB) refers to molecules such as proteins, DNA, RNA, cells, or extracellular vesicles in blood and other bodily fluids that ...

Biobank for Translational Medicine: Standard Operating Procedures for Optimal Sample Management

Biobanks are key research infrastructures aimed at the collection, storage, processing, and sharing of high-quality human biological samples and associated data for research, diagnosis, and personalized medicine. The Biobank for Translational and Digital Medicine Unit at the European Institute of Oncology (IEO) is a landmark in this field. Biobanks collaborate with clinical divisions, internal and external research groups, and industry, supporting patients' treatment and scientific progress, including innovative diagnostics, biomarker discovery, and clinical trial design. Given the central role of biobanks in modern research, biobanking standard operating procedures (SOPs) should be extremely precise. SOPs and controls by certified specialists ensure the highest quality of samples for the implementation of science-based, diagnostic, prognostic, and therapeutic personalized strategies. However, despite numerous efforts to standardize and harmonize biobanks, these protocols, which follow a strict set of rules, quality controls, and guidelines based on ethical and legal principles, are not easily accessible. This paper presents the biobank standard operating procedures of a large cancer center.

Biobanks are crucial resources for biomedical research and the Biobank for Translational and Digital Medicine Unit at the European Institute of Oncology is a model in this field. Here, we provide a detailed description of biobanks' standard operating procedures for the management of different types of human biological samples.

转化医学生物样本库:优化样品管理的标准操作程序

Biobanks are key research infrastructures aimed at the collection, storage, processing, and sharing of high-quality human biological samples and ...

前列腺切除术标本标测和解剖方案

Enhancing Prostate Tumor Biobanking Reliability with Improved Sampling Technique and Histological Characterization

Acquiring fresh and well-characterized tumor tissue samples is critical for conducting high-quality &#34;omics&#34; studies. However, it can be particularly challenging in the context of prostate cancer (PC) due to the unique nature of this organ and the high heterogeneity associated with this tumor. On the other hand, histopathologically characterizing samples before their storage without causing significant tissue alterations is also an intriguing challenge. In this context, we present a new method for acquiring, mapping, characterizing, and micro-dissecting resected prostate tissue based on anatomopathological criteria.
Unlike previously published protocols, this method reduces the time required for histopathological analysis of the prostate specimen without compromising its structure, which is crucial for assessing surgical margins. Furthermore, it enables the delineation and micro-macro dissection of fresh prostate tissue samples, with a focus on histological tumor areas defined by pathological criteria such as Gleason score, precursor lesions (high-grade prostatic intraepithelial neoplasia - PIN), and inflammatory lesions (prostatitis). These samples are then stored in a Biobank for subsequent research analyses.

作者聚焦:通过改进组织取样和生物样本库推进前列腺癌研究

This protocol describes a method for facilitating the collection of samples from radical prostatectomy specimens. The goal is to map, characterize, and micro-macro dissect tissue samples from the specimens based on anatomopathological criteria before storing them in a Biobank.

通过改进采样技术和组织学表征提高前列腺肿瘤生物样本库的可靠性

Acquiring fresh and well-characterized tumor tissue samples is critical for conducting high-quality &#34;omics&#34; studies. However, it can be ...

标准化卵巢癌类器官生物样本库

Strategy for Biobanking of Ovarian Cancer Organoids: Addressing the Interpatient Heterogeneity across Histological Subtypes and Disease Stages

While the establishment of an ovarian cancer biobank from patient-derived organoids along with their clinical background information promises advances in research and patient care, standardization remains a challenge due to the heterogeneity of this lethal malignancy, combined with the inherent complexity of organoid technology. This adaptable protocol provides a systematic framework to realize the full potential of ovarian cancer organoids considering a patient-specific variability of progenitors. By implementing a structured experimental workflow to select optimal culture conditions and seeding methods, with parallel testing of direct 3D seeding versus a 2D/3D route, we obtain, in most cases, robust long-term expanding lines suitable for a broad range of downstream applications. 
Notably, the protocol has been tested and proven efficient in a great number of cases (N = 120) of highly heterogeneous starting material, including high-grade and low-grade ovarian cancer and stages of the disease with primary debulking, recurrent disease, and post-neoadjuvant surgical specimens. Within a low Wnt, high BMP exogenous signaling environment, we observed progenitors being differently susceptible to the activation of the Heregulin 1 &#223; (HER&#223;-1)-pathway, with HER&#223;-1 promoting organoid formation in some while inhibiting it in others. For a subset of the patient's samples, optimal organoid formation and long-term growth necessitate the addition of fibroblast growth factor 10 and R-Spondin 1 to the medium. 
Further, we highlight the critical steps of tissue digestion and progenitor isolation and point to examples where brief cultivation in 2D on plastic is beneficial for subsequent organoid formation in the Basement Membrane Extract type 2 matrix. Overall, optimal biobanking requires systematic testing of all main conditions in parallel to identify an adequate growth environment for individual lines. The protocol also describes the handling procedure for efficient embedding, sectioning, and staining to obtain high-resolution images of organoids, which is required for comprehensive phenotyping.

作者聚焦：推进卵巢癌的个性化医疗 

This protocol offers a systematic framework for the establishment of ovarian cancer organoids from different disease stages and addresses the challenges of patient-specific variability to increase yield and enable robust long-term expansion for subsequent applications. It includes detailed steps for tissue processing, seeding, adjusting media requirements, and immunofluorescence staining.

卵巢癌类器官的生物样本库策略：解决跨组织学亚型和疾病阶段的患者间异质性

While the establishment of an ovarian cancer biobank from patient-derived organoids along with their clinical background information promises advances in ...

建立以临床为基础的生物制剂

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转化医学生物样本库:优化样品管理的标准操作程序

通过改进采样技术和组织学表征提高前列腺肿瘤生物样本库的可靠性

通过改进采样技术和组织学表征提高前列腺肿瘤生物样本库的可靠性

通过改进采样技术和组织学表征提高前列腺肿瘤生物样本库的可靠性

卵巢癌类器官的生物样本库策略：解决跨组织学亚型和疾病阶段的患者间异质性