作者要感谢他的意见，博士，Borsig Lubor（生理研究所，瑞士苏黎世大学）为肺灌注技术。这项工作是支持的Krebsliga的Kanton苏黎世，沃尔特·沃尔夫基金会和Johanna，苏黎世​​，的莉迪亚Hochstrasser基金会，苏黎世，瑞士国家科学基金会，SNF，瑞士法律组成的社团性质的Balgrist Schweizerischer，大学和补助苏黎世。

1。 原位肺灌注和固定<ol><li>麻醉小鼠通过腹腔注射〜150微升磷酸盐缓冲盐水（PBS）含112.5毫克/公斤（体重）氯胺酮，16.5 mg / kg的甲苯噻嗪，和15毫克/千克乙酰丙嗪（或由另一个麻醉含有适当的止痛药）。 </li><li>当反射的鼠标不再观察到，在手术台上修复它背下来，并用70％的乙醇湿润的皮毛光滑的头发下来。 </li><li>打开的颈部和腹部的皮肤，把它的侧面或删除它。 </li><li>打开腹膜和胸部充足的大小。防止大血管破裂（ 如腔jugularis），以避免降低工作效率的后续灌注。 </li><li>切下腔静脉的肝尾下。 </li><li>用20毫升的注射器配备了一个21G - 24G针缓慢注入10〜15毫升PBS进入右心室跳动的心脏，直到肺完全是白色的，心脏停止跳动（心搏停止）。 </li><li>用血管钳掐断肝脏和膜片的上述血管。 </li><li>使用10毫升注射器装有用21G - 24G针注入〜2毫升在PBS中的3％多聚甲醛（PFA）到右心室。 </li><li>揭开气管外的胸部。使用相同的注射器注入〜3毫升3％PFA中进气管在PBS颅（胸廓之外），直到肺膨胀。后立即除去针头，捏关闭气管尾端的穿刺用血管钳，等待10分钟，以允许固定。 </li><li>横切血管上方的血管钳，移除夹具并注入〜2毫升PBS到右心室，以除去过量的PFA。 </li><li>穿刺用针的所有肺叶的下部，取出气管和血管钳在PFA中的第一穿刺，直到进气管尾端嵌入介质/ PBS 1:1注入3-5的溶液PolyFreeze毫升肺裂片被替换的解决方案。 </li><li>小心取出肺拉的心，用钳子和横断结缔组织韧带，血管和气管。保持心脏连接到肺部。 </li><li>根据如何继续进行，肺组织分析继续在第2或3所述的协议。如果你想同时做分析，删除的肺叶并继续在3。处理剩余的连接到心脏的裂片2下所述。 </li></ol> 2。在整个肺叶的lacZ的标签转移性肿瘤细胞的可视化<ol><li>将肺紧闭盖在塑料杯中，并在室温下30-60分钟，用2％甲醛的PBS固定。 </li><li>取出固定的解决方案，并彻底冲洗3次，PBS。 </li><li>加入至少10毫升新鲜制备的5 - 溴-4 - 氯-3 - 吲哚基-β-D-半乳糖苷（X-Gal的）染色溶液（X-Gal的原液[40毫克/毫升二甲基甲酰胺] 1:40稀释的基本染色液，pH值7.1（ 表1），避光）。 </li><li>把一块纱布上的游泳肺保持完全的解决方案，并把只有松散的盖子上锅，让空气交换。 </li><li>在37°C孵育3-5小时，避光。 </li><li>删除X-Gal的解决方案，一次与PBS冲洗，以除去残留的X-Gal的解决方案（可选）和4％PFA。 </li></ol> 3。 lacZ的标签转移性肿瘤细胞在肺癌组织中冰冻切片的可视化<ol><li>充液的标记嵌入模具到1/3，与未稀释的PolyFreeze灌封介质。把肺叶之上，填补了包埋剂，直到完全覆盖叶。尽量避免气泡。 </li><li>嵌入式肺部，在4℃孵育20分钟。 </li><li>然后冻结嵌入式的肺部慢慢在干冰和异戊烷和存储的混合物中，在-80℃下</li><li>切7-10微米低温部分在低温恒温器，将其安装在SuperFrostPlus显微镜幻灯片。 </li><li>立即用的X-Gal染色溶液在37℃下孵育部分在一个加湿室中放置24小时。 </li><li>冲洗5分钟，然后在蒸馏水中短暂的在PBS中2倍的幻灯片。 </li><li>反污渍，核固红为10-30秒，在蒸馏水冲洗干净，并安装IMMU安装的幻灯片。 </li></ol> 4。代表性的成果浅井等报道在原始文章中的邓恩衍生的的LM8 OS鼠标模型，SC原发肿瘤来自父母邓恩细胞的，不同的那些来自高转移LM8子细胞，不自发地形成检测肺转移同系C3H小鼠14。在这里报道的技术，我们的一致性形成转移Dunn/LM8鼠标OS车型。我们利用了稳定LacZ基因转导的邓恩和LM8细胞s和在现场的小鼠肺灌注和固定的协议。 LacZ基因转导的和非转导控制Dunn和LM8细胞在图1中示出的代表图像的灌注和非灌注肺小鼠皮下注射。在小鼠注入与控制邓恩细胞中，宏观和微观转移仍无法检测到非灌注和灌注肺（ 图1A，I-IV）。但是，有趣的是，在唐恩-lacZ的细胞注射小鼠，X-gal染色显示蓝色的微小转移的非灌注肺的表面上（ 图1A，ⅵ）的单细胞或小的细胞簇（&lt;0.1毫米）。原位灌注，固定的肺部，进一步提高了探测邓恩-lacZ的微小转移灶（ 图1A， 八 ）。然而，没有观察到生长到宏观灶（ 图1A，五， 七 ）。/ P&gt; 大于在与对照LM8细胞的，半透明的，几乎检测不到macrometastatic灶小鼠注射的直径为0.1mm，非灌注肺（ 图1B，i）的确认。灌注的肺（ 图1B，ⅲ）未提高病灶的检测。然而，在小鼠注射LM8-lacZ的多个细胞的X-Gal染色蓝色宏（ 图1B，v）和微转移（ 图1B，ⅵ）进行检测的表面上的非灌注器官。此外，灌注肺进一步提高了探测性的宏观和微转移灶（ 图1B， 七 - 八 ）。因此，微型和巨转移成为可见的，主要是由于在更高的密度和较大的数字灶下方的器官表面的半透明，其中灌注组织也成为可见。 一个额外Histological分析，使用肺组织冰冻切片，证实了改进的可探测lacZ基因转导的邓恩和LM8细胞注入小鼠的肺转移。唐恩-lacZ或LM8-lacZ的细胞来自原发性肿瘤的小鼠，确认肺部分（ 图2）不同，在原发肿瘤的小鼠的各自的控制单元，微转移或即使是单细胞灶。此外，巨转移也LM8-lacZ的细胞相比，与对照动物注射LM8细胞（ 图2C，D）注射的小鼠中更清晰可见。 <img alt="图1" src="/files/ftp_upload/4162/4162fig1.jpg" /> 图1。检测自发转移的非转导（I-IV） 和 LacZ转导（V-VIII），邓恩（A）和LM8（B）细胞在非灌注（I，II，V，VI）和灌注（III，IV，VII，VIII）在C3H小鼠肺组织。代表转移的X-GAL-STA独立非执行董事全肺（Ⅴ，ⅦA和B）和各自的特写镜头（VI，VIII中的A和B）显示为蓝色。由星号表示Macrometastatic灶（0.1毫米）的非标记LM8细胞在B（I-IV）注射小鼠的器官。在近距离拍摄中所示的区域代表整个肺部。标尺显示图像全肺的1毫米和0.1毫米特写。 <a href="http://www.jove.com/files/ftp_upload/4162/4162fig1large.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图2" src="/files/ftp_upload/4162/4162fig2.jpg" /> 图2。Dunn和LM8微转移的肺癌冰冻切片检测。 OCT嵌入式肺组织的部分的X-Gal染色溶液中在37℃下孵育24小时，在一个加湿室中，然后用核固红复染。箭头指向微小转移确认唐恩-lacZ的（B）或LM8-lacZ的细胞（D）注射的小鼠的肺切片。中号icrometastases仍然与非转导的邓恩（A）或LM8细胞（C）注入小鼠的肺切片中检测不到。比例尺表明0.1毫米。

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没有利益冲突的声明。

这里提出的结果，在Dunn/LM8鼠标操作系统模型展示的力量，新成立的方法，它结合了X-Gal染色LacZ基因标记的肿瘤细胞的原位灌注/肺组织固定。这两种技术的组合允许微转移病灶的高灵敏度检测到单细胞水平，也提高了肺表面（ 图1），以及在肺部（ 图2）上的可视化的巨转移。虽然在X-Gal染色也允许在其他器官， 原位灌注/固定的（微）转移的检测因为天然的血液和组织相关的颜色只是略有改善肺以外的组织中转移灶的可探测性这些器官13。即使灌注被定向到另一个器官， 例如肝脏，除去血液中仅部分地改善下注的对比度之间的X-Gal染色的天然颜色的器官。然而，该方法适用于任何类型的LacZ基因标记的肿瘤细胞，将显着改善的可探测性肺转移的电平下降到休眠状态的单细胞微转移，使宏观和微转移的一个简单的和可靠的定量。这种方法的一个限制是基于报告基因，包括荧光素酶和荧光蛋白质，和所有其他技术的转基因表达的稳定性。如在图2d所示，不是所有的肿瘤细胞内的macrometastatic灶被染成蓝色，表示缺乏β-半乳糖苷酶的活性。这可能是坏死，但它更可能是由于转基因表达的亏损。我们观察到，邓恩和LM8细胞中的表达lacZ和其他转基因的下调，即使在连续选择是非常有效的。因此，我们在近期的研究中切换到小鼠K12和K7M2和人力居屋，143B和SAOS-2骨肉瘤细胞株，都保持稳定的lacZ的表达在体外以及体内高达100％，随着时间的推移。 一旦保证稳定的lacZ的表达，这种技术可应用于研究基因操作的肿瘤细胞中， 例如 ，机械地调查过程中组织定植13以及用于开发和测试的新的治疗方法，旨在消除转移病灶15 ，16。此外，它可以作为一个基准放射成像技术，如PET，（微）CT和MRI，用于早期发现转移病灶的改善。在最近的PET研究（未出版），不同的示踪剂，我们验证在体内检测到肺转移随后的体外所描述的协议。在一个新的小动物微型CT（SKYSCAN）正在进行的研究是迄今为止AB文件在体内检测肺转移到的尺寸为0.5毫米，下降到0.3毫米的体外 ，但我们的目标是在一个分辨率为0.1毫米。有趣的是，这是宏观微转移的原位灌注，X-Gal染色相结合的方法，我们区分大小的限制。这再次彰显这个简单的和具有成本效益的技术的敏感性和有效性。

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> Narketan10（氯胺酮） </td><td> VétoquinolAG </td><td></td><td> 100毫克/毫升溶液</td></tr><tr><td>赛拉嗪（二甲苯胺噻嗪） </td><td>特莱制药公司</td><td></td><td> 20毫克/毫升溶液</td></tr><tr><td> Prequilan（乙酰丙嗪） </td><td> FATRO SpA的</td><td></td><td> 10毫克/毫升溶液</td></tr><tr><td>斗牛犬型Serrefine血管钳</td><td>精细科学工具</td><td> 18051-35 </td><td>弯曲的，35毫米</td></tr><tr><td>有盖的塑料杯</td><td> Semadeni </td><td> 2988 </td><td> 25毫升有盖杯</td></tr><tr><td> 5 - 溴-4 - 氯-3 - 吲哚基-β-D-半乳糖苷（X-Gal的）;粉末</td><td> AXXORA </td><td> ALX-582-002-G005 </td><td>溶解在N，N-二甲基甲酰胺40毫克/毫升，存储光保护，在-20℃下</td></tr><tr><td>基本染色溶液，平衡至pH为7.1，存储的光保护，在4℃下最大。 1个月</td><td>自制</td><td></td><td> 100 ml / L的10xPBS，1.64 g / L的铁氰化钾，2.1 g / L的亚铁氰化钾（CN）6，2毫升/升1M氯化镁，2毫升/升10％NP40，1毫升/ L的10％的钠 - 脱氧胆酸</td></tr><tr><td> PolyFreeze包埋剂</td><td> Polysciences的瑞士经销商：Brunschwig </td><td> 19636-1 </td><td></td></tr><tr><td>嵌入模具</td><td> Polysciences的</td><td> 18646A-1 </td><td></td></tr><tr><td>徕卡CM1850低温恒温器</td><td>徕卡显微系统</td><td></td><td></td></tr><tr><td> Superfrost防脱载玻片</td><td>门泽尔</td><td> J1800AMN​​Z </td><td></td></tr><tr><td>核固红 - 硫酸铝溶液</td><td>科色度瓦尔德克GMBH＆CO KG区ributor：Medite </td><td> 84-0241-00 </td><td></td></tr><tr><td>免疫山</td><td> Thermo Electron公司，瑞士的分销商：Histocom </td><td> 9990412 </td><td></td></tr></tbody></table> 表1。试剂和设备。

improved visualization of lung metastases at single cell resolution in mice by combined in-situ perfusion of lung tissue and x-gal staining of lacz-tagged tumor cells

转移是主要死亡原因，在大多数类型的癌症，因此在癌症研究的一个主要焦点。然而，影像学和成功在他们的治疗消灭微小转移灶的检测仍然是有限的。 尽管动物模型已经被证明是非常宝贵的工具，用于癌症研究1，监视/微转移灶的可视化仍然是一个挑战和不准确的评价转移扩散的临床前研究，可能导致令人失望的结果在临床试验中2。因此，有极大的兴趣，在改进的方法，最终使重现性好，可靠的检测在正常组织中单个细胞水平上的转移。因此，主要的重点是对技术，它允许在体内的肿瘤细胞的检测，如微计算机断层扫描（微CT），正电子发射断层扫描（PET），生物发光或荧光成像&lt;SUP&gt; 3,4。目前，我们正在优化这些技术在原发肿瘤的生长和转移在不同的骨肉瘤模型体内监测。其中的一些技术也可以用于体外分析转移旁边类似的qPCR 5，流式细胞仪6或不同类型的组织学染色的经典方法。为基准，在本研究中，我们已经建立的稳定转染或转导的肿瘤细胞与 lacZ基因编码细菌的酶β-半乳糖苷酶代谢的显色底物5 -溴-4 -氯-3 -吲哚基-β-D -吡喃半乳糖苷（X-Gal的）一种不溶性靛兰染料7和允许高灵敏度和选择性的组织化学蓝染色的肿瘤细胞在小鼠组织体外单细胞水平，如下所示。这是一个低成本，而不是大量设备的工具，它允许转移8在研究的精确验证评估新ticancer疗法9-11。 X-gal染色的一个限制因素是低的对比度，以及血管化组织， 例如血液相关的红色染色。在肺组织中，这个问题是可以解决的原位灌注肺，Borsig 等12灌注小鼠的肺部麻醉下，他们从血液中清除，修复和嵌入在原地 ，这是最近成立的一个技术通过气管通胀。此方法可以防止肺崩溃，从而保持形态的功能，肺泡，从而提高了质量的组织进行组织学分析。在本研究中，我们描述了一种新的协议，它充分利用了X-gal染色的 lacZ表达的肿瘤细胞的结合， 在原位灌注，肺组织固定。这家精致的协议允许高灵敏度检测的细胞在肺单发转移，使我们在最近的研究为detect“冬眠”肺微转移的小鼠模型13，原本是描述非转移性14。

Watch this Scientific Journal Video about Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tum

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

在本研究中报道的新颖协议在小鼠经合并在单细胞分辨率允许选择性检测肺转移<em&gt;在原位</em&gt;肺灌注固定和X-Gal染色<em&gt; lacZ的</em&gt;标记的肿瘤细胞。

肺转移瘤在小鼠体内的组合在单细胞分辨率的可视化改进<em&gt;在原位</em灌注肺组织和X-gal染色<em&gt; lacZ的</em&gt;标记的肿瘤细胞

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of ...

improved-visualization-lung-metastases-at-single-cell-resolution-mice

Research

JoVE Journal

Medicine

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

17.2K 次观看.  Balgrist University Hospital, Zurich. 在本研究中报道的新颖协议在小鼠经合并在单细胞分辨率允许选择性检测肺转移在原位肺灌注固定和X-Gal染色 lacZ的标记的肿瘤细胞。

视频: Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and &#8220;pearls of wisdom&#8221;/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

的隔离方法<em&gt;离体</em&gt;肺灌注的大鼠模型：教训制定鼠EVLP课程中学

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

科研

医学

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered genotypes now possess the ability to invade and survive in other tissues. In this protocol, mouse mammary tumors are removed and primary cells are prepared from tumors. The cells isolated from this procedure are then available for gene profiling experiments. For successful metastasis, these cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system. The lungs are the typical target of breast cancer metastasis. A set of genes have been discovered that mediates the selectivity of metastasis to the lung. Here we describe a method of studying lung metastasis from a genetically engineered mouse model.. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. MEF cells from the same animal type provide a clue of non-cancer cell gene expression. Together, these techniques are useful in studying mouse mammary tumorigenesis, its associated signaling mechanisms and pathways of the abnormalities in embryos.

The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

原发灶和MEF细胞分离，研究肺转移

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

评价肺转移的小鼠乳腺肿瘤模型定量实时荧光定量PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute respiratory distress syndrome (ARDS), characterized by neutrophilic alveolitis, and injury to both alveolar epithelium and vascular endothelium. Clinically, ARDS is defined by acute onset of hypoxemia, bilateral patchy pulmonary infiltrates and non-cardiogenic pulmonary edema. Human studies have provided us with valuable information about the physiological and inflammatory changes in the lung caused by ARDS, which has led to various hypotheses about the underling mechanisms. Unfortunately, difficulties determining the etiology of ARDS, as well as a wide range of pathophysiology have resulted in a lack of critical information that could be useful in developing therapeutic strategies.
Translational animal models are valuable when their pathogenesis and pathophysiology accurately reproduce a concept proven in both in vitro and clinical settings. Although large animal models (e.g., sheep) share characteristics of the anatomy of human trachea-bronchial tree, murine models provide a host of other advantages including: low cost; short reproductive cycle lending itself to greater data acquisition; a well understood immunologic system; and a well characterized genome leading to the availability of a variety of gene deletion and transgenic strains. A robust model of low pH induced ARDS requires a murine ALI that targets mainly the alveolar epithelium, secondarily the vascular endothelium, as well as the small airways leading to the alveoli. Furthermore, a reproducible injury with wide differences between different injurious and non-injurious insults is important.
The murine gastric acid aspiration model presented here using hydrochloric acid employs an open tracheostomy and recreates a pathogenic scenario that reproduces the low pH pneumonitis injury in humans. Additionally, this model can be used to examine interaction of a low pH insult with other pulmonary injurious entities (e.g., food particles, pathogenic bacteria).

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

急性肺损伤开放气管切开胃酸吸入小鼠模型结果在急性最大非致命性肺损伤

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

Generation of Human 3D Lung Tissue Cultures (3D-LTCs) for Disease Modeling

Translation of novel discoveries to human disease is limited by the availability of human tissue-based models of disease. Precision-cut lung slices (PCLS) used as 3D lung tissue cultures (3D-LTCs) represent an elegant and biologically highly relevant 3D cell culture model, which highly resemble in situ tissue due to their complexity, biomechanics and molecular composition. Tissue slicing is widely applied in various animal models. 3D-LTCs derived from human PCLS can be used to analyze responses to novel drugs, which might further help to better understand the mechanisms and functional effects of drugs in human tissue. The preparation of PCLS from surgically resected lung tissue samples of patients, who experienced lung lobectomy, increases the accessibility of diseased and peritumoral tissue. Here, we describe a detailed protocol for the generation of human PCLS from surgically resected soft-elastic patient lung tissue. Agarose was introduced into the bronchoalveolar space of the resectates, thus preserving lung structure and increasing the tissue&#39;s stiffness, which is crucial for subsequent slicing. 500 &#181;m thick slices were prepared from the tissue block with a vibratome. Biopsy punches taken from PCLS ensure comparable tissue sample sizes and further increase the amount of tissue samples. The generated lung tissue cultures can be applied in a variety of studies in human lung biology, including the pathophysiology and mechanisms of different diseases, such as fibrotic processes at its best at (sub-)cellular levels. The highest benefit of the 3D-LTC ex vivo model is its close representation of the in situ human lung in respect of 3D tissue architecture, cell type diversity and lung anatomy as well as the potential for assessment of tissue from individual patients, which is relevant to further develop novel strategies for precision medicine.

Generation of Human 3D Lung Tissue Cultures 3D-LTCs for Disease Modeling

Here, we present a protocol for the preparation of agarose-filled human precision-cut lung slices from resected patient tissue that are suitable for generating 3D lung tissue cultures to model human lung diseases in biological and biomedical studies.

用于疾病建模的人三维肺组织培养 (3D-LTCs) 的生成

Translation of novel discoveries to human disease is limited by the availability of human tissue-based models of disease. Precision-cut lung slices (PCLS) ...

Quantitative Analysis of Cellular Composition in Advanced Atherosclerotic Lesions of Smooth Muscle Cell Lineage-Tracing Mice

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel interventions have remained elusive. This is likely due, in part, to a reliance on preclinical prevention models investigating the effects of genetic manipulations or pharmacological treatments on atherosclerosis development rather than the established disease. Also, results of these studies are often confounding because of the use of superficial lesion analyses and a lack of characterization of lesion cell populations. To help overcome these translational hurdles, we propose an increased reliance on intervention models that employ investigation of changes in cellular composition at a single cell level by immunofluorescent staining and confocal microscopy. To this end, we describe a protocol for testing a putative therapeutic agent in a murine intervention model including a systematic approach for animal dissection, embedding, sectioning, staining, and quantification of brachiocephalic artery lesions. In addition, due to the phenotypic diversity of cells within late-stage atherosclerotic lesions, we describe the importance of using cell-specific, inducible lineage tracing mouse systems and how this can be leveraged for unbiased characterization of atherosclerotic lesion cell populations. Together, these strategies may assist vascular biologists to more accurately model therapeutic interventions and analyze atherosclerotic disease and will hopefully translate into a higher rate of success in clinical trials.

We propose a standardized protocol to characterize the cellular composition of late-stage murine atherosclerotic lesions including systematic methods of animal dissection, tissue embedding, sectioning, staining, and analysis of brachiocephalic arteries from atheroprone smooth muscle cell lineage tracing mice.

平滑肌细胞近系追踪小鼠晚期动脉粥样硬化病变细胞成分的定量分析

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel ...

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. Various approaches have been described, such as the inhalation of aerosolized LPS as well as nasal or intratracheal instillation. The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by direct intratracheal LPS instillation and perform FACS analysis of blood samples, bronchoalveolar lavage (BAL) fluid, and lung tissue. After intraperitoneal sedation, the trachea is exposed and LPS is administered via a 22 G venous catheter. A robust and reproducible inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier is induced within hours to days, depending on the LPS dosage used. Collection of blood samples, BAL fluid, and lung harvesting, as well as the processing for FACS analysis, are described in detail in the protocol. Although the use of the sterile LPS is not suitable to study pharmacologic interventions in infectious diseases, the described approach offers minimal invasiveness, simple handling, and good reproducibility to answer mechanistic immunological questions. Furthermore, dose titration as well as the use of alternative LPS preparations or mouse strains allow modulation of the clinical effects, which can exhibit different degrees of ALI severity or early vs. late onset of disease symptoms.

Presented here is a step-by-step procedure to induce acute lung injury in mice by direct intratracheal lipopolysaccharide instillation and to perform FACS analysis of blood samples, bronchoalveolar lavage fluid, and lung tissue. Minimal invasiveness, simple handling, good reproducibility, and titration of disease severity are advantages of this approach.

通过直接内切内脂多糖灌输诱导小鼠急性肺损伤

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism

Lung transplantation (LTx) remains the standard of care for end-stage lung disease. A shortage of suitable donor organs and concerns over donor organ quality exacerbated by excessive geographic transportation distance and stringent donor organ acceptance criteria pose limitations to current LTx efforts. Ex situ lung perfusion (ESLP) is an innovative technology that has shown promise in attenuating these limitations. The physiologic ventilation and perfusion of the lungs outside of the inflammatory milieu of the donor body affords ESLP several advantages over traditional cold static preservation (CSP). There is evidence that negative pressure ventilation (NPV) ESLP is superior to positive pressure ventilation (PPV) ESLP, with PPV inducing more significant ventilator-induced lung injury, pro-inflammatory cytokine production, pulmonary edema, and bullae formation. The NPV advantage is perhaps due to the homogenous distribution of intrathoracic pressure across the entire lung surface. The clinical safety and feasibility of a custom NPV-ESLP device have been demonstrated in a recent clinical trial involving extender criteria donor (ECD) human lungs. Herein, the use of this custom device is described in a juvenile porcine model of normothermic NPV-ESLP over a 12 h duration, paying particular attention to management techniques. Pre-surgical preparation, including ESLP software initialization, priming, and de-airing of the ESLP circuit, and the addition of anti-thrombotic, anti-microbial, and anti-inflammatory agents, is specified. The intraoperative techniques of central line insertion, lung biopsy, exsanguination, blood collection, cardiectomy, and pneumonectomy are described. Furthermore, particular focus is paid to anesthetic considerations, with anesthesia induction, maintenance, and dynamic modifications outlined. The protocol also specifies the custom device's initialization, maintenance, and termination of perfusion and ventilation. Dynamic organ management techniques, including alterations in ventilation and metabolic parameters to optimize organ function, are thoroughly described. Finally, the physiological and metabolic assessment of lung function is characterized and depicted in the representative results.

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism

This paper describes a porcine model of negative pressure ventilation ex situ lung perfusion, including procurement, attachment, and management on the custom-made platform. Focus is made on anesthetic and surgical techniques, as well as troubleshooting.

非原位负压通气肺灌注：肺功能和代谢的评估

Lung transplantation (LTx) remains the standard of care for end-stage lung disease. A shortage of suitable donor organs and concerns over donor organ ...

Lung Rapid Recovery Procurement Combined with Abdominal Normothermic Regional Perfusion in Controlled Donation after Circulatory Death

Controlled donation after circulatory death (cDCD) has contributed to increasing donor numbers all over the world. Experiences published in the last years confirm that the outcomes after lung transplantation from cDCD are similar to those from brain death donors; however, the utilization of lungs from asystole donors remains low. Several reasons may be involved: different legal frameworks among countries and centers with different premortem interventions, inadequate lung donor care before procurement, or even poor experience with cDCD procedures and protocols.
Initially, the rapid recovery technique was commonly employed for the procurement of thoracic and abdominal organs in cDCD, but, in the last decade, abdominal normothermic regional perfusion (ANRP) with extracorporeal membrane oxygenation devices has become a useful method to restore blood flow to abdominal organs, allowing their quality improvement and their functional assessment prior to transplantation. This makes the donation procedure more complex and generates doubts about injury to the grafts due to dual temperature.
The aim of this article is to describe a protocol based on a single center experience with Maastricht III donors combining lung cooling rapid recovery in the thorax and abdominal normothermic regional perfusion. Tips and tricks focused on premortem interventions and lung procurement procedure techniques are explained. This may help to minimize the reluctance among professionals to use this combined technique and encourage other donor centers to use it, despite the increased complexity of the procedure.

The protocol combines a lung cooling rapid recovery technique with abdominal normothermic regional perfusion for abdominal graft procurement in controlled asystole donors, which is a safe and useful method to expand the donor pool.

肺快速恢复采购联合腹部正常区域灌注在循环死亡后受控捐献中的应用

Controlled donation after circulatory death (cDCD) has contributed to increasing donor numbers all over the world. Experiences published in the last years ...

使用琼脂糖包埋的振动切片机进行创新的肺部成像

Automated Vibratome Sectioning of Agarose-Embedded Lung Tissue for Multiplex Fluorescence Imaging

Due to its inherent structural fragility, the lung is regarded as one of the more difficult tissues to process for microscopic readouts. To add structural support for sectioning, pieces of lung tissue are commonly embedded in paraffin or OCT compound and cut with a microtome or cryostat, respectively. A more recent technique, known as precision-cut lung slices, adds structural support to fresh lung tissue through agarose infiltration and provides a platform to maintain primary lung tissue in culture. However, due to epitope masking and tissue distortion, none of these techniques adequately lend themselves to the development of reproducible advanced light imaging readouts that would be compatible across multiple antibodies and species. 
To this end, we have developed a tissue-processing pipeline, which utilizes agarose embedding of fixed lung tissue, coupled to automated vibratome sectioning. This facilitated the generation of lung sections from 200 &#181;m to 70 &#181;m thick, in mouse, pig, and human lungs, which require no antigen retrieval, and represent the least "processed" version of the native isolated tissue. Using these slices, we reveal a multiplex imaging readout capable of generating high-resolution images whose spatial protein expression can be used to quantify and better understand the mechanisms underlying lung injury and regeneration.

作者聚焦：使用琼脂糖包埋的肺组织切片研究空间蛋白表达 

We have developed a tissue-processing technique utilizing a vibratome and agarose-embedded lung tissue to generate lung sections, thereby permitting the acquisition of high-resolution images of lung architecture. We employed immunofluorescence staining to observe spatial protein expression using specific lung structural markers.

用于多重荧光成像的琼脂糖包埋肺组织的自动振动切片机切片

Due to its inherent structural fragility, the lung is regarded as one of the more difficult tissues to process for microscopic readouts. To add structural ...