Name: pancreatic tissue dissection to isolate viable single cells
Uploaded: 2023-05-26T13:00:00
Description: Watch this Scientific Journal Video about Pancreatic Tissue Dissection to Isolate Viable Single Cells at JoVE.com

我们要感谢 Avital Sarusi-Portuguez 博士在数据分析方面的帮助，以及 Dror Kolodkin-Gal 博士在之前的研究中为建立协议提供的帮助。我们感谢Parnas实验室的所有过去和现在的成员。我们感谢 Gillian Kay 博士和 Michael Kanovsky 博士在编辑方面的帮助。该项目已获得以色列科学基金会资助（第 526/18 O.P.号）、Alex U. Soyka 计划和以色列癌症研究基金（研究职业发展奖）的资助。

希伯来大学（以色列耶路撒冷）和哈达萨医学中心（以色列耶路撒冷）的联合伦理委员会（机构动物护理和使用委员会）批准了动物福利研究方案（MD-18-15417-5“小鼠胰腺癌的组织动力学”），此处介绍的方案符合动物试验和研究的所有相关伦理法规。希伯来大学是国际实验动物护理评估和认证协会认可的机构。
注：小鼠品系原液 #007908、原液 #019378 和原液 #008179 是从杰克逊?...

<ol>
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D., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics,+2019.">Cancer statistics, 2019.</a> CA: a Cancer Journal for Clinicians. 69 (1), 7-34 (2019).</li><li>Yachida, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distant+metastasis+occurs+late+during+the+genetic+evolution+of+pancreatic+cancer.">Distant metastasis occurs late during the genetic evolution of pancreatic cancer.</a> Nature. 467 (7319), 1114-1117 (2010).</li><li>Peng, 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=Author+correction:+single-cell+RNA-seq+highlights+intra-tumoral+heterogeneity+and+malignant+progression+in+pancreatic+ductal+adenocarcinoma.">Author correction: single-cell RNA-seq highlights intra-tumoral heterogeneity and malignant progression in pancreatic ductal adenocarcinoma.</a> Cell Research. 29 (9), 777 (2019).</li><li>Hruban, R. H., Wilentz, R. E., Kern, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+progression+in+the+pancreatic+ducts.">Genetic progression in the pancreatic ducts.</a> The American Journal of Pathology. 156 (6), 1821-1825 (2000).</li><li>Schlesinger, 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=Single-cell+transcriptomes+of+pancreatic+preinvasive+lesions+and+cancer+reveal+acinar+metaplastic+cells'+heterogeneity.">Single-cell transcriptomes of pancreatic preinvasive lesions and cancer reveal acinar metaplastic cells' heterogeneity.</a> Nature Communications. 11 (1), 4516 (2020).</li><li>Kolodkin-Gal, 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=Senolytic+elimination+of+Cox2-expressing+senescent+cells+inhibits+the+growth+of+premalignant+pancreatic+lesions.">Senolytic elimination of Cox2-expressing senescent cells inhibits the growth of premalignant pancreatic lesions.</a> Gut. 71 (2), 345-355 (2021).</li><li>Kopp, J. 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=Identification+of+Sox9-dependent+acinar-to-ductal+reprogramming+as+the+principal+mechanism+for+initiation+of+pancreatic+ductal+adenocarcinoma.">Identification of Sox9-dependent acinar-to-ductal reprogramming as the principal mechanism for initiation of pancreatic ductal adenocarcinoma.</a> Cancer Cell. 22 (6), 737-750 (2012).</li><li>Elyada, 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=Cross-species+single-cell+analysis+of+pancreatic+ductal+adenocarcinoma+reveals+antigen-presenting+cancer-associated+fibroblasts.">Cross-species single-cell analysis of pancreatic ductal adenocarcinoma reveals antigen-presenting cancer-associated fibroblasts.</a> Cancer Discovery. 9 (8), 1102-1123 (2019).</li><li>Bernard, V., 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=Single-cell+transcriptomics+of+pancreatic+cancer+precursors+demonstrates+epithelial+and+microenvironmental+heterogeneity+as+an+early+event+in+neoplastic+progression.">Single-cell transcriptomics of pancreatic cancer precursors demonstrates epithelial and microenvironmental heterogeneity as an early event in neoplastic progression.</a> Clinical Cancer Research. 25 (7), 2194-2205 (2019).</li><li>Moncada, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+microarray-based+spatial+transcriptomics+and+single-cell+RNA-seq+reveals+tissue+architecture+in+pancreatic+ductal+adenocarcinomas.">Integrating microarray-based spatial transcriptomics and single-cell RNA-seq reveals tissue architecture in pancreatic ductal adenocarcinomas.</a> Nature Biotechnology. 38 (3), 333-342 (2020).</li><li>Hwang, W. 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在本文中，我们提出了胰腺组织解离的方案。该方案简单易用，并提供了一种在恶性肿瘤过程（包括实体瘤）的不同阶段从胰腺组织中分离活单细胞的工具。在以前的研究中，不同类型的胶原酶被用来消化胰腺8,9。使用非常有效的胶原酶，如胶原酶D，可产生大量的免疫细胞和较低比例的上皮细胞。使用胶原酶P可以进行胰腺消化，适用于完整或肿瘤胰腺...

胰腺导管腺癌 （PDAC） 是最具侵袭性的癌症类型之一1.临床证据支持这样一种观点，即PDAC是由KRAS原癌基因2突变驱动的外分泌系统细胞（包括腺泡细胞）发展而来的。
胰腺肿瘤包括许多不同的细胞类型，并且已经证明恶性细胞仅占肿瘤质量的 20%-50%3。不同的细胞类型与上皮细胞相互作用，支持其转化，并促进肿瘤的形成和生长。早期事件导致腺泡化生，从而引起称为胰腺上皮内瘤变 （PanINs） 的微观病变，在某些情况下可发展为 PDAC4。
迫切需要研究这些相互作用并瞄准关键信号。单细胞RNA测序（scRNA-seq）是一种强大的方法，它以单细胞分辨率揭示基因表达，从而跟踪上皮细胞所经历的变化，从而能够探索胰腺癌的发展。 
组织解剖和消化为单细胞是scRNA-seq实验的第一阶段。有几个因素使胰腺组织消化特别具有挑战性：i）腺泡细胞占胰腺的90%以上，腺泡细胞含有大量的消化酶，包括蛋白酶和RNase，这些酶会降低基于RNA的文库的质量;（ii） 腺泡细胞非常敏感，如果使用标准方案，可能会裂解;（iii） 腺泡细胞以非常高的水平表达少量基因。因此，如果这些细胞在实验过程中裂解，可能会污染观察到的其他细胞的基因表达谱;（iv） 从肿瘤中恢复的胰腺组织是结缔组织增生的，因此很难在不损伤细胞的情况下进行解剖。因此，尽管需要保持所有细胞类型的高活力，但腺泡细胞的大量和敏感性增加了额外的复杂性。这些因素给实现单细胞悬液带来了困难，该悬浮液的存活率超过80%，并且没有团块，这是scRNA-seq实验所需的。 
在这里，我们开发了一种使用胰蛋白酶C和胶原酶P的方案，以及频繁的组织监测。这支持解离为单细胞，同时保持高活力以支持 scRNA-seq 实验的成功 5,6。

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pancreatic tissue dissection to isolate viable single cells

胰腺包括两个主要系统：产生和分泌激素的内分泌系统，以及约占胰腺90%的外分泌系统，包括产生和分泌消化酶的细胞。消化酶在胰腺腺泡细胞中产生，储存在称为酶原的囊泡中，然后 通过 胰管释放到十二指肠中以启动代谢过程。腺泡细胞产生的酶可以杀死细胞或降解游离RNA。此外，腺泡细胞很脆弱，常见的解离方案会导致大量死细胞和游离蛋白酶和RNase。因此，胰腺组织消化的最大挑战之一是恢复完整和活的细胞，尤其是腺泡细胞。本文中介绍的协议显示了我们为满足这一需求而开发的两步方法。该方案可用于消化正常胰腺、包含癌前病变的胰腺或包含大量基质和免疫细胞的胰腺肿瘤。

在最近发表的工作5中，我们应用上述协议来探索使用小鼠模型的PDAC开发的早期阶段。小鼠经过基因工程改造，包括 Ptf1a-CreER、LSL-Kras-G12D、LSL-tdTomato 7 盒，其允许在他莫昔芬注射后腺泡细胞中表达组成型活性 KRAS。 
颈椎脱位后（根据小鼠伦理方案），切除胰腺，并应用上述方案（见 图1</stro...

Watch this Scientific Journal Video about Pancreatic Tissue Dissection to Isolate Viable Single Cells at JoVE.com

Pancreatic Tissue Dissection to Isolate Viable Single Cells

胰腺化生细胞是引起胰腺肿瘤的恶性细胞的前体。然而，分离完整的活胰腺细胞具有挑战性。在这里，我们提出了一种有效的胰腺组织解离方法。然后，这些细胞可用于单细胞RNA测序（scRNA-seq）或二维或三维共培养。

胰腺组织解剖以分离活的单细胞

The pancreas includes two major systems: the endocrine system, which produces and secretes hormones, and the exocrine system, which accounts for ...

所证明的胰腺组织解离方案很简单，并提供了一种在恶性肿瘤过程（包括实体瘤）的不同阶段从胰腺组织中分离活单细胞的工具。恢复完整和活的腺泡细胞是一项重大挑战。该方案可以从正常胰腺、具有癌前病变的胰腺或含有大量基质和免疫细胞的胰腺肿瘤中恢复活的单细胞。使用该方案解剖人类胰腺肿瘤或发炎的胰腺可以帮助研究这些疾病以寻找新的生物标志物和治疗方法。该方法易于使用，只需最少的练习即可提供所需的结果。该视频将有助于查看协议的小细节。在开始解剖之前，将所有仪器和设备准备好在冰上。固定安乐死的小鼠后，用70%乙醇喷洒其腹部。用剪刀和镊子在动物的生殖器区域做一个2.5厘米的V形切口，然后向上完全打开腹腔。将胃定位在小鼠的左侧和胰腺的左侧，胰腺靠近脾脏。使用两个镊子，将胰腺与胃和十二指肠分开，不要撕裂。继续将胰腺与小肠、空肠和回肠分开。将胰腺向右侧移动，用镊子分离胰腺与胸腔之间的剩余连接，使胰腺和附着的脾脏完全分离。小心地取出不含肠系膜脂肪或其他邻近组织的胰腺，并将其摊开在冰上的培养皿中进行检查。按照文中提到的组合物，制备解离缓冲液1和2，提前洗涤缓冲液和酶活性终止液。将胰腺置于冰上的50毫升管中，并用10%胎牛血清（FBS）和汉克平衡盐溶液（HBSS）冲洗。脂肪会漂浮，胰腺会下沉。这是一种可视化和快速去除仍附着在胰腺上的污染性白色脂肪组织的简单方法。接下来，将小鼠胰腺组织转移并保存在含有5毫升HBSS的无菌培养皿中，直至切割。使用Noyes剪刀和手术刀，将胰腺切成一到三立方毫米的小块。将组织转移到离心管中后，以 350G 和 4 摄氏度离心 5 分钟。吸出并弃去上清液以除去细胞碎片和血细胞。在37摄氏度下搅拌，将碎片重悬于含有0.02%胰蛋白酶-C和0.05%EDTA的解离缓冲液1中10分钟。立即用 10% FBS 和 Dulbecco 改良的 Eagle 培养基 （DMEM） 洗涤，并在 350G 和 4 摄氏度下离心 5 分钟。通过将沉淀重悬于10毫升洗涤缓冲液中再次洗涤，并像以前一样在下一个解离步骤之前离心5分钟。将胰腺在解离缓冲液 2 中在 37 摄氏度下孵育 15 分钟，以每分钟 180 转的速度搅拌。15分钟后，使用无菌血清移液管上下剧烈移液胰腺碎片10次，进行机械解离。重复使用尺寸递减的移液器，从 25、10 到 5 毫升。孵育样品，使其达到 37 摄氏度。使用光学显微镜根据悬浮液中单细胞的量监测解离。使用台盼蓝监测细胞活力。继续孵育，每五分钟取样一次检查解离。孵育至90%的细胞分离成单个细胞。胰腺组织充分解离后，表现为胰腺碎片消失和溶液浑浊度增加，用酶活性终止液在4摄氏度下洗涤5分钟两次，停止酶促反应。从这一步开始，将细胞悬浮液保持在冰上。将细胞悬液通过70微米尼龙网。较小的尼龙网尺寸可能会降低细胞活力。在显微镜下检查细胞活力。在计数细胞之前，重悬并用 5 至 10 毫升冰冷的缓冲洗涤溶液洗涤沉淀。如果观察到几个红细胞，则在室温下用红细胞裂解缓冲液处理细胞两分钟。在观察到团块的情况下，如前所述，应再次用胰蛋白酶处理样品。如果活力低于 80%，则应使用磁激活细胞分选或带有 MACS MS 色谱柱的 MACS 死细胞去除试剂盒分离活细胞。分离的胰腺组织的红色是由于腺泡细胞中tdTomato的表达所致。在解离的早期阶段，分离的细胞数量很少，团块数量很大。后来，在避免减少活细胞数量的同时获得分离的活细胞。较长的孵育时间降低了细胞的活力。使用荧光激活的细胞分选估计 tdTomato 阳性细胞。温和解离方案支持多种细胞类型的回收，具有高活力，可以对所需细胞类型进行细胞分选。使用血细胞计数器进行活细胞到死细胞计数。冷冻胰腺切片的荧光图像显示tdTomato阳性腺泡细胞。单细胞RNA测序证实了在所有时间点的每个切除组织中检测到荧光激活细胞分选中检测到的所有细胞类型。研究了腺泡细胞中羧肽酶1或CPA1的表达，表明对其他细胞类型转录组的污染最小。需要注意的是，较长的孵育时间会降低细胞的活力，因此在显微镜下每隔几分钟监测样品并观察组织解离和细胞活力非常重要。细胞分离方案可用于单细胞RNA测序、培养细胞或作为类器官的起始原料。该技术可以探索胰腺癌如何发展，并研究浸润发炎或癌组织细胞之间的相互作用。

Research

JoVE Journal

Cancer Research

Capture and Release of Viable Circulating Tumor Cells from Blood

捕获和血液中的活循环肿瘤细胞释放

We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip ...

科研

癌症研究

Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts

3维球形共培养胰腺癌细胞和成纤维细胞的制备及代谢试验

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated ...

A Syngeneic Pancreatic Cancer Mouse Model to Study the Effects of Irreversible Electroporation

自体胰腺癌小鼠模型研究不可逆电穿孔的影响

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches ...

Isolation of Proximal Fluids to Investigate the Tumor Microenvironment of Pancreatic Adenocarcinoma

分离近端液研究胰腺腺癌肿瘤微环境

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables ...

Generation of an Orthotopic Xenograft of Pancreatic Cancer Cells by Ultrasound-Guided Injection

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant ...

Syngeneic Mouse Orthotopic Allografts to Model Pancreatic Cancer

同系小鼠原位同种异体移植物模拟胰腺癌

Pancreatic ductal adenocarcinoma (PDAC) is a very complex disease characterized by a heterogeneous tumor microenvironment made up of a diverse stroma, ...

Quantifying Telomere Length Using Non-Radioactive Chemiluminescence Detection

Optimization of Performance Parameters of the TAGGG Telomere Length Assay

Author Spotlight: Optimization of Performance Parameters of the TAGGG Telomere Length Assay  

TAGGG端粒长度测定性能参数优化

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening ...

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Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts from Fresh Tissue

Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue

Author Spotlight: Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue  

从新鲜组织中建立胰腺癌来源的肿瘤类器官和成纤维细胞

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. ...

3D Technique to Visualize Mitochondrial Cristae in Pancreatic Cancer Cells

A Three-Dimensional Technique for the Visualization of Mitochondrial Ultrastructural Changes in Pancreatic Cancer Cells

Author Spotlight: A Three-Dimensional Technique for the Visualization of Mitochondrial Ultrastructural Changes in Pancreatic Cancer Cells  

一种可视化胰腺癌细胞线粒体超微结构变化的三维技术

Understanding the dynamic features of the cell organelle ultrastructure, which is not only rich in unknown information but also sophisticated from a ...

Reprogramming PDAC and Pancreatic Cells into Induced Pluripotent Stem Cells

Reprogramming Pancreatic Ductal Adenocarcinoma to Pluripotency

Author Spotlight: Reprogramming Cancer Cells to iPSCs to Study Disease Progression and Treatment Targets

将胰腺导管腺癌重编程为多能性

The generation of induced pluripotent stem cells (iPSCs) using transcription factors has been achieved from almost any differentiated cell type and has ...