我们想感谢Hesham Abdelbary博士为图2中的数据提供人类手术标本。

1。组织处理<ol><li>为了达到最佳效果，本议定书应使用新鲜分离的组织在DMEM媒体含10％FBS，1％Pennicilin /链霉素溶液，0.1％Amphothericin B溶液立即手术后加工前交存。如果这是不可能的，组织可以留过夜，4度加工之前，在这个媒介。 </li><li>处理样品之前，至少5分钟，他们存放在一个70％的乙醇溶液消毒金属钳和一次性刀片。还准备用2毫升含10％FBS，1％Pennicilin /链霉素溶液，0.1％Amphothericin B.的DMEM媒体24孔板</li><li>在层流罩细胞培养，使用消毒镊子收集组织样本，并存入一个空15厘米的培养皿中的组织，保持侧面的盖子，无菌的一面。 </li><li>在细胞培养罩，使用2毫米活检冲床，获得的各种组织内的区域，如图1不同的内核。存款与产钳的帮助下15厘米的培养皿的盖子的核心，在每个核心之间留下足够的空间，使他们可以很容易地沿水平轴切。 </li><li>分割成4甚至用消毒的刀片，如图1季度每个核心。 </li><li>使用吸管尖，在不同的井，从A1核心每个核心季度为A4，如图1中，已经包含了1.5毫升的DMEM含10％FBS，1％Pennicilin /链霉素溶液，0.1％Amphothericin B液。重复每个核心。这应使肿瘤的代表性抽样，同时尽量减少在一个给定的井/条件的偏见。为了更好的代表性，增加核心的数量。 </li></ol> 2。组织可行性评估<ol><li>以下组织处理，加25μlAlamar蓝以及＃A1＃A2的如图1所示，并与5％CO2培养箱在37度孵育1小时。 </li><li>以下孵化alamar蓝色，删除3倍，从每个A1和A2 100μL，转移到一个96孔板不同井。使用荧光酶标仪（530励磁，590排放）读取信号，并记录您的数据。 </li><li> Alamar蓝信号已被阅读后，转移组织的所有作品，用吸管尖，从孔A1至C1包含的DMEM，10％FBS + PS + AmphoB。要小心，不要过量的媒体传输孔A1。 </li><li>感染井A3和A4分别为10 6 PFU的绿色荧光蛋白表达和荧光素酶表达的牛痘病毒在25μL媒体稀释。 A2可以用，根本没有表示任何特定的基因，包括病毒感染。 </li><li> 72小时后，加入25μLAlamar蓝井C1和D1，并重复步骤2.2 </li></ol> 3。 GFP转基因表达的荧光显微镜可视化<ol><li>删除所有的细胞培养介质覆盖的组织块，以良好的荧光图片。 </li><li>使用荧光功能的解剖镜下，先取相衬图像，在一个适当的决议</li><li>切换到荧光模式，并使用适当的过滤器，可视化在这种情况下，绿色荧光蛋白的转基因利益，拍照。 </li><li>用另一种波长的利益（如RFP）的转基因尽量可能的一个图片的背景荧光的荧光过滤器</li></ol> 4。荧光素酶表达的可视化使用活体成像系统（ IVIS） <ol><li>确保IVIS是初始化，然后再开始。 </li><li>在井A4和B4，添加5μL荧光素底物10毫克/毫升，拌匀5分钟，在室温孵育。 </li><li> IVIS曝光时间设置为5秒，您的井的图片。如果图像是饱和的，重复使用较低的曝光时间。如果没有信号，增加曝光时间。 </li><li>使用IVIS成像软件，您可能会去除背景，使用以及B4的信号，并选择地区的利益，以量化的发光信号。 </li></ol> 5。评估斑块检测病毒滴度<ol><li>量化病毒滴度在此之前，组织必须先匀浆释放病毒颗粒。这可以在数个月后，受感染的组织样本保存于-80 </li><li>称取样品，需要使用分析规模匀浆。在这种情况下，我们将使用收集的样本以及A2。 </li><li>将组织5毫升聚苯乙烯圆底的Falcon管，并添加1毫升的PBS。使用组织匀浆器匀浆的组织。如果有必要，储存于-80匀浆病毒滴度在稍后的时间评估。 </li><li>滴度牛痘病毒，第一盘1万U2OS细胞在6孔板，在37度孵育过夜，5％的CO 2潮湿ified孵化器，他们已达到约95％汇合翌日。 </li><li>使用无血清媒体做系列稀释的病毒，一定要更改之间的每个稀释步骤的提示。通常情况下，我们在10稀释1 100μL转移到900μL。如何作出许多稀释取决于病毒的预期收益率。 </li><li>使稀释后，除去覆盖镀U20S细胞，然后加入500μL稀释病毒的股票（每个稀释度）感染U2OS细胞的媒体。在37摄氏度度在5％ 二氧化碳培养箱中孵育1小时分钟的细胞。 </li><li>在此期间，热身2 ×集中的DMEM含20％胎牛血清，以及3％的CMC溶液在37度的热水浴。 </li><li> 1小时的潜伏期后，覆盖感染U2OS细胞取出介质。 1:1卷3％CMC：2X的DMEM，20％胎牛血清混合在一起，这种混合使用2毫升涵盖每个受感染的细胞U2OS以及。 </li><li>细胞放置在一个37度的5％二氧化碳培养箱48小时。 </li><li> 48小时后，每口井的CMC覆盖上添加2毫升的甲醇 - 冰醋酸固定液，在室温下孵育10分钟在细胞培养罩。 </li><li>丢弃固定覆盖和清洗使用自来水井的其余。 </li><li> 2毫升每一个考马斯亮蓝溶液染色固定U2OS细胞，孵育30分钟，在室温在低速板振动筛。 </li><li>删除从井的考马斯染色，并用自来水冲洗板。允许干用了大约一个小时的盖子关闭。 </li><li>由此产生的病毒斑块可以很容易地在图2c的可视化。板可以无限期地储存在这个阶段。 </li><li>在10和100之间的斑块是可见的稀释步骤计数斑块。 </li><li>乘以使用稀释计算斑块的数量乘以2得到的数字，给在PFU / ml的滴度。例如，如果计算在百万倍稀释25斑块，初始未经稀释的样品的效价乘以25乘以2或50万PFU / ml的1万元。进一步的体重除以这个数字最初测量样本报告PFU / G滴度</li></ol> 6。代表性的成果： 为了准确地确定手术获得的正常/肿瘤组织样本是否是或不是病毒infectable，首先必须确保组织样本，至少是可行的。图1A显示了使用一个新陈代谢的染料（Alamar蓝色），超过了至少72小时内能保持正常和肿瘤组织代谢活跃，可行性进行评估。这表明，组织培养离体可以支持病毒复制。溶瘤病毒的一个显着优点是，它们可以被设计来表达治疗或成像转基因。图2D - E显示绿色荧光蛋白或荧光素酶可以从感染体外组织检测，进一步支持这些组织是可行的，也infectable。虽然转基因表达的增加一般与病毒复制有关，并不一定等同于生产病毒的生命周期，导致自我放大和传播，这被认为是治疗活动的重要。出于这个原因，它是必要的，以确定是否比是用于最初感染的组织产生更多的病毒。图2b显示斑块检测病毒量化后，更多的病毒感染后72小时获得，然后感染后，立即组织收集。总体而言，这些数据表明，手术切除的肿瘤组织可以继续生存在细胞培养至少72小时内，在这段时间内病毒的复制，可以支持。 <img src="/files/ftp_upload/2854/2854fig1.jpg" alt="图1"/> 图1。概述组织取样/切片协议。组织样本中删除了2毫米，随后分成四个季度，这是随机分布成井A1 - A4的分为核心处理。颜色代码显示在每口井所用试剂。灰色阴影的广场内的水井，每季度从6个人的核心描绘。孔A1的组织与媒体转移到一个新的井（C1）后初步alamar蓝色阅读。二读的是在72小时与病毒潜伏期结束。韦尔斯A4和B4是辅以荧光素后的72小时孵化后感染<img src="/files/ftp_upload/2854/2854fig2.jpg" alt="图2"/> 图2。生存能力和病人的组织样本infectability 。 一）组织活力alamar蓝色信号，在2小时和72小时后收集测量。 Y轴表示的空白校正alamar蓝色信号，B）牛痘病毒滴度每克2和72小时后感染C）牛痘病毒斑例如U2OS细胞，考马斯染色，D）发光信号从病人维维荧光素酶受感染的组织取得的组织样本收集的影像使用 IVIS E）荧光显微镜图片VV - GFP感染的病人组织样本。

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	<li>Parato, K. A., Senger, D., Forsyth, P. A., Bell, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progress+in+the+battle+between+oncolytic+viruses+and+cancer.">Recent progress in the battle between oncolytic viruses and cancer.</a> Nat Rev Cancer. 5, 965-976 (2005).</li><li>Renouf, L. C., Thway, K., Sibtain, A., McNeish, I. A., Newbold, K. L., Goldsweig, H., Coffin, R., Nutting, C. 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在这个协议中的关键步骤之一是获得新鲜的组织样本。如果样品到细胞培养在一个不合适的介质（如PBS）的手术室在漫长的等待之后，这可能会危及组织的生存能力和防止infectability。值得注意的是，正常组织本质上更容易出现这些影响比肿瘤组织。另一个关键的一点是有多少个核用于样品的组织和它们的大小的一致性。大小不一致会导致跨患者样本的可变性因素，如组织缺氧和infectectable表面会因为波动的核心和核心宿舍的大小而异。虽然这可以部分解决利用组织切片，这里介绍的方法的一个优势是，它是相对比较容易，不易污染，广泛适用于多种组织类型，包括软或粘性组织，这是不容易适合组织切片。值得注意的是，核心的数量可以增加，以获得更好的组织的代表。此外，内核可以被切成多块（如5-8），以适应其他潜在的检测，如DNA，RNA或蛋白质的提取并行完成。然而，在该内核可裁剪重现大小的块的数量将取决于实际大小的核心，它可以通过使用不同大小的corers，修改。可选的一种方式，以帮助获得再现大小的组织块甚至核心之前，用一把尺子进一步细分成小块的长度。该协议自然可以被修改，以适应其他病毒，我们发现，组织可以是可行的，并支持长达6天的复制。该协议可以进一步扩展到其他维拉利表达外源基因，包括cytrokines测量。感染后，也可以组织石蜡包埋进一步切片染色及免疫组织化学方法，允许进一步细化组织的组织学，它如何与病毒感染10-12有关。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td>高糖DMEM </td><td> Hyclone公司</td><td> SH30243.01 </td><td></td></tr><tr><td>胎牛血清</td><td> NorthBio公司</td><td> NBSF - 701 </td><td></td></tr><tr><td> Amphothericin乙解决方案</td><td>西格玛爱秩序</td><td> A2942 </td><td>使用0.1％ </td></tr><tr><td> Pennicilin链霉素</td><td>西格玛爱秩序</td><td> P4333 </td><td>使用1％ </td></tr><tr><td> Alamar蓝</td><td> Invitrogen公司</td><td> DAL1025 </td><td></td></tr><tr><td> D -荧光素钾盐</td><td>分子影像产品</td><td> D -荧光素钾盐1G </td><td>在PBS重悬在10毫克/毫升和过滤0.22微米过滤器</td></tr><tr><td> MEM粉末</td><td> Gibco公司</td><td> ＃41500018 </td><td>请在建议量的一半，使0.22微米过滤器的两倍MEM和过滤器在使用之前</td></tr><tr><td>羧甲基纤维素钠（CMC）， </td><td>西格玛爱秩序</td><td> C9481 </td><td>请在3％溶液用去离子水和高压灭菌。请注意，粉可能需要一些时间，以重悬</td></tr><tr><td>考马斯亮蓝R </td><td>西格玛爱秩序</td><td> B7920 </td><td></td></tr><tr><td> 2毫米活检冲床</td><td> Miltex </td><td> MX - 33 - 31 </td><td></td></tr><tr><td>双刃准备叶片</td><td> Personna医疗保健</td><td> 74-0002 </td><td></td></tr><tr><td>荧光disection显微镜</td><td>徕卡</td><td>模型M205 FA </td><td></td></tr><tr><td> 在活体成像系统（IVIS） </td><td>卡尺生命科学</td><td> IVIS ® 200系列</td><td></td></tr><tr><td>组织Tearor </td><td> Biospec产品</td><td>模型985370-395 </td><td></td></tr></tbody></table>

ex vivo infection of live tissue with oncolytic viruses

溶瘤病毒（OVS）选择性复制和杀灭肿瘤细胞的1的新型疗法。多种，包括单纯疱疹病毒，呼肠孤病毒，牛痘OVS作为癌症治疗的溶瘤平台的有效性进行评估的临床试验目前正在进行2-5。溶瘤病毒的主要特点之一是它们可以被转基因快报记者转基因，这使得它能够以可视化的显微镜或生物发光成像 6,7组织感染。这提供了一个独特的优势，因为它是可能的感染治疗之前， 患者体内组织，以确定的溶瘤病毒治疗成功 8的可能性。为此，关键是适当样本，以弥补组织的异质性和评估组织的生存能力，特别是前9感染的组织。同样重要的是按照病毒的复制，记者转基因溶瘤平台以及以下的同质化，以歧视胎死腹中和生产力之间的感染组织的直接滴定表示。本议定书的目的是要解决这些问题，本文介绍1。细胞培养2肿瘤组织取样和准备。该组织的可行性评估，使用的代谢染料alamar蓝色3。体外感染牛痘病毒的培养组织表达GFP或萤火虫荧光素酶4。转基因表达的检测，荧光显微镜或使用活体成像系统（IVIS） 5。斑块的检测病毒的定量。这种全面的方法提出了几个优点，包括便于组织处理，组织异质性的补偿，控制组织活力，和流产的感染和骨真正的病毒复制之间的歧视。

Watch this Scientific Journal Video about Ex Vivo Infection of Live Tissue with Oncolytic Viruses at JoVE.com

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

溶瘤病毒是有希望的癌症治疗。的能力，以确定的活组织标本从患者治疗前infectability，是一个独特的优势，这种治疗方法。本协议描述了如何处理组织<em&gt;体外</em&gt;溶瘤病毒和随后的病毒定量的感染。

前溶瘤病毒的活组织的体内感染

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness ...

ex-vivo-infection-of-live-tissue-with-oncolytic-viruses

Research

JoVE Journal

Medicine

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

12.5K 次观看.  Ottawa Hospital Research Institute (OHRI). 溶瘤病毒是有希望的癌症治疗。的能力，以确定的活组织标本从患者治疗前infectability，是一个独特的优势，这种治疗方法。本协议描述了如何处理组织体外溶瘤病毒和随后的病毒定量的感染。

视频: Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Ex Vivo Culture of Patient Tissue &amp; Examination of Gene Delivery

This video describes the use of patient tissue as an ex vivo model for the study of gene delivery. Fresh patient tissue obtained at the time of surgery is sliced and maintained in culture. The ex vivo model system allows for the physical delivery of genes into intact patient tissue and gene expression is analysed by bioluminescence imaging using the IVIS detection system. The bioluminescent detection system demonstrates rapid and accurate quantification of gene expression within individual slices without the need for tissue sacrifice. This slice tissue culture system may be used in a variety of tissue types including normal and malignant tissue and allows us to study the effects of the heterogeneous nature of intact tissue and the high degree of variability between individual patients. This model system could be used in certain situations as an alternative to animal models and as a complementary preclinical mode prior to entering clinical trial.

Ex Vivo Culture of Patient Tissue &amp; Examination of Gene Delivery

This article describes the culture of patient tissue slices for gene delivery studies and subsequent analysis of gene expression using IVIS bioluminescence imaging.

患者组织及基因传递考试前的体外培养

This video describes the use of patient tissue as an ex vivo model for the study of gene delivery.  Fresh patient tissue obtained at the time of surgery ...

科研

医学

Handling of the Cotton Rat in Studies for the Pre-clinical Evaluation of Oncolytic Viruses

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact. For this strategy to be successful, recent studies have shown that involvement of the host immune system is essential. Therefore, oncolytic virotherapy should be evaluated within the context of an immunocompetent model. Furthermore, the study of antitumor therapies in tolerized animal models may better recapitulate results seen in clinical trials. Cotton rats, commonly used to study respiratory viruses, are an attractive model to study oncolytic virotherapy as syngeneic models of mammary carcinoma and osteosarcoma are well established. However, there is a lack of published information on the proper handling procedure for these highly excitable rodents. The handling and capture approach outlined minimizes animal stress to facilitate experimentation. This technique hinges upon the ability of the researcher to keep calm during handling and perform procedures in a timely fashion. Finally, we describe how to prepare cotton rat mammary tumor cells for consistent subcutaneous tumor formation, and how to perform intratumoral and intraperitoneal injections. These methods can be applied to a wide range of studies furthering the development of the cotton rat as a relevant pre-clinical model to study antitumor therapy.

Cotton rats are extremely excitable and have a strong flight-or-fight response. A handling method optimized to reduce the stress of the animals is described which will make cotton rats more accessible as a preclinical model.

处理棉鼠的研究为溶瘤病毒的临床前评估

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact.  For this strategy to be ...

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular processes that govern metastatic dissemination and growth are still elusive. Live imaging allows visualization of the dynamic and spatial interactions of cells and their microenvironment. Solid tumors commonly metastasize to the lungs. However, the anatomical location of the lungs poses a challenge to intravital imaging. This protocol provides a relatively simple and quick method for ex vivo live imaging of the dynamic interactions between tumor cells and their surrounding stroma within lung metastasis. Using this method, the motility of cancer cells as well as interactions between cancer cells and stromal cells in their microenvironment can be visualized in real time for several hours. By using transgenic fluorescent reporter mice, a fluorescent cell line, injectable fluorescently labeled molecules and/or antibodies, multiple components of the lung microenvironment can be visualized, such as blood vessels and immune cells. To image the different cell types, a spinning disk confocal microscope that allows long-term continuous imaging with rapid, four-color image acquisition has been used. Time-lapse movies compiled from images collected over multiple positions and focal planes show interactions between live metastatic and immune cells for at least 4 hr. This technique can be further used to test chemotherapy or targeted therapy. Moreover, this method could be adapted for the study of other lung-related pathologies that may affect the lung microenvironment.

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

We describe a relatively simple method for ex vivo live imaging of the tumor cell-stroma interactions within lung metastasis, utilizing fluorescent reporters in mice. Using spinning-disk confocal microscopy, this technique enables visualization of live cells for at least 4 hr and could be adapted to study other inflammatory lung conditions.

体外肺转移及微环境的实时成像

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular ...

Transarterial Administration of Oncolytic Viruses for Locoregional Therapy of Orthotopic HCC in Rats

Hepatocellular carcinoma (HCC) is a disease with limited treatment options and poor prognosis. In recent years, oncolytic virotherapies have proven themselves to be potentially powerful tools to fight malignancy. Due to the unique dual blood supply in the liver, it is possible to apply therapies locally to orthotopic liver tumors, which are predominantly fed by arterial blood flow. We have previously demonstrated that hepatic arterial delivery of oncolytic viruses results in safe and efficient transduction efficiency of multifocal HCC lesions, resulting in significant prolongation of survival in immune competent rats. This procedure closely mimics the application of transarterial embolization in patients, which is the standard palliative care provided to many HCC patients. The ability to administer tumor therapies through the hepatic artery in rats allows for a highly sophisticated preclinical model for evaluating novel viral vectors under development. Here we describe the detailed protocol for microdissection of the hepatic artery for infusion of oncolytic virus vectors to treat orthotopic HCC.

Oncolytic virotherapies are under development as novel therapeutics for the treatment of hepatocellular carcinoma (HCC). Here we describe a method for locoregional therapy of HCC via hepatic arterial administration of oncolytic virus.

溶瘤病毒的Transarterial管理局大鼠原位肝癌的局部治疗

Hepatocellular carcinoma (HCC) is a disease with limited treatment options and poor prognosis.  In recent years, oncolytic virotherapies have proven ...

Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation

Human small intestinal enteroids are derived from the crypts and when grown in a stem cell niche contain all of the epithelial cell types. The ability to establish human enteroid ex vivo culture systems are important to model intestinal pathophysiology and to study the particular cellular responses involved. In recent years, enteroids from mice and humans are being cultured, passaged, and banked away for future use in several laboratories across the world. This enteroid platform can be used to test the effects of various treatments and drugs and what effects are exerted on different cell types in the intestine. Here, a protocol for establishing primary stem cell-derived small intestinal enteroids derived from neonatal mice and premature human intestine is provided. Moreover, this enteroid culture system was utilized to test the effects of species-specific breast milk. Mouse breast milk can be obtained efficiently using a modified human breast pump and expressed mouse milk can then be used for further research experiments. We now demonstrate the effects of expressed mouse, human, and donor breast milk on the growth and proliferation of enteroids derived from neonatal mice or premature human small intestine.

Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation

This protocol describes how to establish an enteroid culture system from neonatal mouse or premature human intestine as well as an efficient method to collect milk from mice.

母乳促进 Enteroids 的生长: 一个前体细胞增殖模型

Human small intestinal enteroids are derived from the crypts and when grown in a stem cell niche contain all of the epithelial cell types. The ability to ...

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. Therefore, adjuvants with both humoral and cellular immuno-stimulatory capabilities that are able to promote cytotoxic T lymphocytes (CTL) responses are urgently needed. Thus, faithful monitoring of adjuvant candidates that induce cross-priming and subsequently enhance CTL generation represents a crucial step in vaccine development. In here we present an application for a method that uses SIINFEKL-specific (OT-I) T cells to monitor the cross-presentation of the model antigen ovalbumin (OVA) in vivo in the presence of different adjuvant candidates. This method represents a rapid test to select adjuvants with the best cross-priming capabilities. The proliferation of CD8+ T cells is the most valuable indication of cross-priming and it is also regarded as a correlate of adjuvant-induced cross-presentation. This feature can be evaluated in different immune organs like lymph nodes and spleen. The extent of the CTL generation can also be monitored, thereby giving insights on the nature of a local (draining lymph node mainly) or a systemic response (distant lymph nodes and/or spleen). This technique further allows multiple modifications for testing drugs that can inhibit specific cross-presentation pathways and also offers the possibility to be used in different strains of conventional and genetically modified mice. In summary, the application that we present here will be useful for vaccine laboratories in industry or academia that develop or modify chemical adjuvants for vaccine research and development.

Rapid In Vivo Assessment of Adjuvant&#039;s Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

We present here an application for a standard immunological technique (CFSE stained OT-I proliferation) intended to rapidly monitor adjuvant-mediated cytotoxic T lymphocyte (CTL) generation in vivo. This fast estimation of CTL capacities is useful for the development of prophylactic vaccines against intracellular pathogens as well as therapeutic cancer vaccines.

疫苗开发中佐剂细胞毒性 T 淋巴细胞生成能力的快速体内评价

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. ...

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, normothermic ex vivo liver perfusion (NEVLP) has been introduced as a method to evaluate and modify organ function. NEVLP has many advantages in comparison to hypothermic and subnormothermic perfusion including reduced preservation injury, restoration of normal organ function under physiologic conditions, assessment of organ performance, and as a platform for organ repair, remodeling, and modification. Both murine and porcine NEVLP models have been described. We demonstrate a rat model of NEVLP and use this model to show one of its important applications &#8212; the use of a therapeutic molecule added to liver perfusate. Catalase is an endogenous reactive oxygen species (ROS) scavenger and has been demonstrated to decrease ischemia-reperfusion in the eye, brain, and lung. Pegylation has been shown to target catalase to the endothelium. Here, we added pegylated-catalase (PEG-CAT) to the base perfusate and demonstrated its ability to mitigate liver preservation injury. An advantage of our rodent NEVLP model is that it is inexpensive in comparison to larger animal models. A limitation of this study is that it does not currently include post-perfusion liver transplantation. Therefore, prediction of the function of the organ post-transplantation cannot be made with certainty. However, the rat liver transplant model is well established and certainly could be used in conjunction with this model. In conclusion, we have demonstrated an inexpensive, simple, easily replicable NEVLP model using rats. Applications of this model can include testing novel perfusates and perfusate additives, testing software designed for organ evaluation, and experiments designed to repair organs.

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant liver donor shortage, and criteria for liver donors have been expanded. Normothermic ex vivo liver perfusion (NEVLP) has been developed to evaluate and modify organ function. This study demonstrates a rat model of NEVLP and tests the ability of pegylated-catalase, to mitigate liver preservation injury.

体外常温肝灌注的小动物模型

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, ...

An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No current animal models of diabetes and oxygen-induced retinopathy develop the full-range progressive changes manifested in human proliferative diabetic retinopathy (PDR). Therefore, understanding of the disease pathogenesis and pathophysiology has relied largely on the use of histological sections and vitreous samples in approaches that only provide steady-state information on the involved pathogenic factors. Increasing evidence indicates that dynamic cell-cell and cell-extracellular matrix (ECM) interactions in the context of three-dimensional (3D) microenvironments are essential for the mechanistic and functional studies towards the development of new treatment strategies. Therefore, we hypothesized that the pathological fibrovascular tissue surgically excised from eyes with PDR could be utilized to reliably unravel the cellular and molecular mechanisms of this devastating disease and to test the potential for novel clinical interventions. Towards this end, we developed a novel method for 3D ex vivo culture of surgically-excised patient-derived fibrovascular tissue (FT), which will serve as a relevant model of human PDR pathophysiology. The FTs are dissected into explants and embedded in fibrin matrix for ex vivo culture and 3D characterization. Whole-mount immunofluorescence of the native FTs and end-point cultures allows thorough investigation of tissue composition and multicellular processes, highlighting the importance of 3D tissue-level characterization for uncovering relevant features of PDR pathophysiology. This model will allow the simultaneous assessment of molecular mechanisms, cellular/tissue processes and treatment responses in the complex context of dynamic biochemical and physical interactions within the PDR tissue architecture and microenvironment. Since this model recapitulates PDR pathophysiology, it will also be amenable for testing or developing new treatments.

Here, we present a protocol to study the pathophysiology of proliferative diabetic retinopathy by using patient-derived, surgically-excised, fibrovascular tissues for three-dimensional native tissue characterization and ex vivo culture. This ex vivo culture model is also amenable for testing or developing new treatments.

增殖性糖尿病视网膜病变纤维血管并发症的体外组织培养模型

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No ...

Immunofluorescence Labelling of Human and Murine Neutrophil Extracellular Traps in Paraffin-Embedded Tissue

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They mature in the bone marrow and are released into the peripheral blood, where they circulate for about 6&#8722;8 h; however, in tissue, they can survive for days. By diapedesis through the endothelium, they leave the blood stream, enter tissues, and migrate towards the site of an infection following chemotactic gradients.&#160;Neutrophils can combat invading microorganisms by phagocytosis,&#160;degranulation, and generation of neutrophil extracellular traps (NETs). This protocol will help to detect NETs in paraffin-embedded tissue. NETs are the result of a process called NETosis, which leads to the release of nuclear, granular, and cytoplasmic components either from living (vital NETosis) or dying (suicidal NETosis) neutrophils. In vitro, NETs form cloud-like structures, which occupy a space several times larger than that of the cells from which they descended. The backbone of NETs is chromatin, to which a selection of proteins and peptides originating from granules and cytoplasm are bound. Thereby, a high local concentration of toxic compounds is maintained so that NETs can capture and inactivate a variety of pathogens including bacteria, fungi, viruses, and parasites, while diffusion of the highly active NET components leading to damage in neighboring tissue is limited. Nevertheless, in recent years it has become apparent that NETs, if generated in abundance or cleared insufficiently, do have pathological potential ranging from autoimmune diseases to cancer. Thus, detection of NETs in tissue samples may have diagnostic significance, and the detection of NETs in diseased tissue can influence the treatment of patients. Since paraffin-embedded tissue samples are the standard specimen used for pathological analysis, it was sought to establish a protocol for fluorescent staining of NET components in paraffin-embedded tissue using commercially available antibodies.

Neutrophil extracellular traps (NETs) are three-dimensional structures generated by stimulated neutrophil granulocytes. It has become clear in recent years that NETs are involved in a wide variety of diseases. Detection of NETs in tissue may have diagnostic relevance, so standardized protocols for labelling NET components are required.

石蜡内嵌组织中人与鼠中性粒细胞外陷阱的免疫荧光标记

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They ...

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to larger animals. Yet, larger mammals are better suited for translational research questions related to normal cardiac physiology, pathophysiology, and preclinical testing of therapeutic agents. To overcome the technical barriers associated with employing a larger animal model in cardiac research, we describe an approach to measure physiological parameters in an isolated, Langendorff-perfused piglet heart. This approach combines two powerful experimental tools to evaluate the state of the heart: electrophysiology (EP) study and simultaneous optical mapping of transmembrane voltage and intracellular calcium using parameter sensitive dyes (RH237, Rhod2-AM). The described methodologies are well suited for translational studies investigating the cardiac conduction system, alterations in action potential morphology, calcium handling, excitation-contraction coupling and the incidence of cardiac alternans or arrhythmias.

The objective of this study was to establish a method for investigating cardiac dynamics using a translational animal model. The described experimental approach incorporates dual-emission optocardiography in conjunction with an electrophysiological study to assess electrical activity in an isolated, intact porcine heart model.

前维沃·兰根多夫注入心脏的心电生理学研究

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to ...