研究以预防失明实验孝行长崎的协助下，这项研究符合眼科及视觉研究中使用动物的ARVO声明。 KJW支持由美国国立卫生研究院资助5T32EY013933和5T32DK007647-20。 VBM是由美​​国国立卫生研究院授予K08EY020530支持。

1。视网膜下注射组装设备<ol><li>购买或制作100微米直径的针从一个玻璃毛细管。这可以手动完成使用萨特P-97的移液管的牵拉或其他类似设备。将被加热的毛细管的端部和拉出，直到达到所希望的直径（100微米）。的较小直径的针可用于基因治疗的载体，但是，这是无损伤的细胞或眼睛的推荐直径为细胞注射。玻璃毛细管针钢针相比，具有非常细的，但钝头，用于可视化的喷射流体和访问到视网膜下的空间，以允许不造成视网膜破损。如果需要使用70％的乙醇，并存储在一个无菌的容器，清洁这些拉针。一个15厘米的无菌组织培养皿用胶带按住针到位是一个方式，以保持注射针的覆盖和在无菌环境中。在这一点上，并发症ETE的设置内的手术室，其中该过程将发生。这是最简单，保持手术室，以便在任何时候都保持无菌状态，尤其是当这个过程完成屏障设施内的所有设备。 </li><li>吸干硅氧烷到注射针和弹出硅氧烷留下沿针的内壁涂层。硅氧烷涂层最大化通过针孔的细胞和病毒，否则会粘的内玻璃毛细管壁的通道。 </li><li>在切割25¾轨距与路厄氏锁定的点设置的采血针后直接（从管中取出针）获取一个约8厘米的无菌塑料管的长度。 </li><li>填写1毫升的子-Q 26 5/8-gauge滑小费用无菌生理盐水的注射器从注射器取下针头。 </li><li>将注射针的塑料管的切断端和在另一附加的注射器的路厄氏锁定结束。 </li><li>吸干无菌生理盐水以填补的死空间在油管和毛细管的针，喷射一些盐水通过针，以确保它被正确地连接无任何泄漏。接着，吸一个小的，大约1微升的量的空气，进入注射针的前端。这将分离的盐水从喷射流体，并提供一个表示结束的喷射流体在外科手术过程的视觉线索。的注射装置应保持在清洁的表面，在手术过程中，用70％乙醇擦拭。此外，在外科手术过程中使用的所有设备应之前和之后，用70％乙醇清洗，应该已经被保持无菌并准备使用的注射针的异常。这些针应设置的手术后，而不是重新使用。 </li><li>最后，吸出含有所需的包装的基因治疗的病毒载体的细胞或干细胞的注射液。这可以通过接受注射溶液的无菌移液管尖端，然后移液无菌细胞培养皿的表面上，该溶液。解决方案现在可以很容易地使用的注射器的注射针吸入。所需的量会有所不同，这取决于对鼠标的年龄。我们的目的是产后5天的小鼠，可注射注射液0.5〜0.8μl的。可以使用较少量，0.5微升或更小，可用于年轻小鼠和较大的量，如1微升，对成年小鼠。病毒滴度或需要确定基于对病毒/细胞被注入，和他们的期望的目标组织的细胞数，因为太多的病毒颗粒或细胞在一个小体积，可导致视网膜细胞毒性。在视频中，可视化的目的，我们已用1.5微升，更大的染料量超过必要注入眼睛。 </li></ol> 2。进入视网膜下腔<ol><li>在无菌手术室，稳定或麻醉的小鼠根据当地批准的的动物处理协议，这将不同年龄的鼠标。常见的麻醉成年小鼠包括异氟醚的使用气体或腹膜内注射0.1 ml/10g体重氯胺酮（100毫克/毫升）和甲苯噻嗪（20毫克/毫升）在无菌生理盐水。对于围产期小鼠，常见的麻醉技术包括使用异氟醚的气体或cryoanesthesia（低温）。所有的麻醉技术，必须由您当地的IACUC委员会批准后方可使用。我们通常使用产后一天（P）5只小鼠，虽然类似的技术可以应用到小鼠年轻P0或成年小鼠。外科手术之前，不能开始鼠标停止移动四肢，不再响应从头到脚捏的。围产期小鼠，他们会变得明显，颜色较浅的血流量会有所放缓，知道，他们是在完全麻醉状态下的另一个关键。 </li><li>在围产期小鼠，使用"维纳斯"直剪刀米AKE约1.5毫米的切口，沿封闭盖裂。小心切的凹口处，自然会打开，映入眼帘的未来。这将确保盖子愈合和正常打开，鼠标的发展过程中。可以用于此目的的一种外科刀片，虽然，我们优选Vannas剪刀，因为它们减少了在此过程中的删余的眼睛的风险。在视频中，用剪刀直接穿刺和切沿睑缘。在学习这门技术，钳，应使用，提起眼睑切割前和减少损害眼睛的风险。 </li><li>拉开眼睑到proptose的眼睛，下方的眼睛保持它proptosed注射用的捏住上眼皮稍微弯曲敷料钳。如果在步骤2中的盖的切口过大时，眼睛将下盖滑回，并防止在注射过程中的适当曝光。对成年小鼠，眼部周围施加轻微的压力与博士埃辛的镊子可以保持的注射眼proptosed。 </li><li>做一个小的巩膜切口或后地球的赤道15度的显微叶片表面划伤。此切口应只有足够大，通过该注射针的前端，但不大于。如果愿意，一个小的，如针灸针针可以用来做切口进入视网膜下腔，而不是显微叶片。在深色的动物，将成为暗棕色色素组织（脉络膜视网膜色素上皮细胞）的切口的点处可见。在更轻的着色或白化小鼠中，可以应用于手术标记笔的显微刀片的前端指示的切口部位离开墨斑。如果明确（玻璃）从切口处的流体回流，刀片放在太深，进入玻璃体腔和治疗病毒或细胞可以进入玻璃体内注射过程中的空间。对于visualizati的目的，我们做了切口，在视频这一点。但是它仍然是可能的，在这种情况下，完成一个视网膜下注射，视网膜脱离引起的喷射流体的存在下，可能无法完全愈合在这种鼠标。因此，必须小心削减注射只眼的外表面，而不是通过在视网膜之前。 </li><li>小心地将注射针插入切口部位和推进平行的外眼壁进入视网膜下腔。推进太接近的外壁或眼内太深，将指示错误的位置注入。理解的针是否是在正确的位置，最好的方法是实践的技术。轻轻色素沉着或白化小鼠的练习，可以帮助进行精确的注射到视网膜下腔。这是因为针和注射流体的这些眼睛内是可见的。 </li><li>轻轻的压力注射器的柱塞和杜松子酒注射所需的流体。如果正确地放置在视网膜下腔穿刺针，温和的背压会被感觉到。如果针头内的玻璃体，将有显着更少的背压和在注射过程中的一些潜在的流体回流。如果有明显的反压，保持针取出针管慢慢前至少一个额外的15秒。这将允许高眼压的均衡，而不是注射的病毒或细胞回流回退的切口部位。可能被注入到视网膜下的空间，由于在注射过程中所需的压力增加的部分的空气和盐水。应注意不要过度注入空气或生理盐水，因为它可以冲洗掉眼注入病毒或细胞。从存在太多视网膜下的空间注入流体，也有可能会造成永久性的视网膜脱离。可视化的目的，多余的染料和注入空气，整数Ø在视频中视网膜下腔。的染料会被刷新退了出去，如果过多的视网膜下腔注入，可以清楚地看到，而且气泡保持在视网膜下的空间并没有消失，进入玻璃体。这是用来强调的是，这些注射剂的眼睛都在正确的位置，所以可以想像针应如何进入切口部位。所有注射到视网膜下的空间会导致一个临时的注射流体的存在下，由于视网膜脱离。如果注射过程正确完成，此临时视网膜脱离会在24小时内和小鼠愈合可用于所有的进一步的实验。大约一个星期，由于外科手术的眼睛可能保持柔软，所以，这一段时间期间应避免第二次注射。 </li><li>新生小鼠，用弯曲的敷料镊子轻轻地按压眼球的后面的盖子，并进入预定轨道。它可能是有用等待一分钟，以便让病毒定居在视网膜下的空间，而不是导致其被刷新的切口部位时，轻推眼进入轨道。手术后，一滴0.5％布比卡因（腔投与局部麻醉药 - 麻佳因）从一个25号针头在无菌生​​理盐水稀释至0.25％可以被放置在注射部位。遇险的小鼠没有明显的迹象显示视网膜手术后，已经有足够的前三年使用布比卡因作为一种长效的局部止痛。的小鼠应始终被监控在手术后寻找任何迹象的困扰，如感染，肿胀，或减少在日常活动的鼠标。如果这些情况发生，应通知兽医和作为腹腔内注射，如丁丙诺啡（0.05毫克/千克），可以进一步的止痛药。 </li><li>根据当地批准的动物处理协议的小鼠从麻醉中醒来。鼠标加热P上应保持广告，以维持正常的体温，因为它从麻醉中醒来。手术过程应小于5分钟即可完成，不计时间接受麻醉的小鼠。因此，加热垫是没有必要在实际过程中，而一个学习的技术，因为该过程可能需要较长的持续时间，但可能需要。不应该被放置在鼠标到一个干净的笼子里，直到它已开始上移动自己的。新生儿，柔和的脚趾掐会帮助他们完全从麻醉中恢复过来，他们应该重新被放回到与母亲的粉红色，运动前。随着时间的推移，应监测小鼠前面列出的任何迹象的困扰。 </li></ol>

<ol>
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没有利益冲突的声明。

此视频技术提供视网膜下注射外科手术成功完成，并且确保被放置在所需的位置，有效地治疗眼科疾病的基因治疗载体或干细胞的说明。这种技术允许RPE或光感受器如视网膜细胞的定位，因为它的基因治疗载体或干细胞衍生的组织放置在这些细胞的周边。以前的方法，其中流体被放置在玻璃体腔的玻璃体内注射，而且必须通过视网膜移植，以针对这些特定领域。玻璃体内注射可降低转导的基因治?...

<table border="1"><tbody><tr><td> 名称 </td><td> 公司 </td><td> 目录 </td><td> 评论 </td></tr><tr><td> 0.8-1.10×100毫米的毛细管（玻璃） </td><td> ，金布尔玻璃公司</td><td> 34502 99 </td><td></td></tr><tr><td>烈焰红唇/褐色微管牵引机</td><td>萨特仪器</td><td> P-97 </td><td>成茂microforge可以用来代替。产品编号MF-900 </td></tr><tr><td> Sigmacote </td><td> Sigma Aldrich公司</td><td> SL2-25ML </td><td>有机硅</td></tr><tr><td> Dubecco的磷酸盐缓冲液，氯化钙和氯化镁</td><td> GIBCO-Invitrogen公司</td><td> 14040-133 </td><td></td></tr><tr><td>安全乐25 3/4G×12";采血车集</td><td> BD真空采血管</td><td> 367298 </td><td></td></tr><tr><td> 1毫升子-Q 265/8G防滑提示注射器</td><td>贝克顿 - 迪金森</td><td> 309597 </td><td></td></tr><tr><td> 0.5-10微升Finnpipette II音量可调移液器</td><td> Fisherbrand </td><td> 21-377-815 </td><td></td></tr><tr><td> 1-200微升自然斜角提示</td><td>美国科技公司</td><td> 1111至1700年</td><td></td></tr><tr><td> Discovery.V8体视显微镜</td><td>蔡司</td><td> MC1500 </td><td></td></tr><tr><td> 60毫米×15毫米风格处理聚苯乙烯细胞培养皿</td><td>康宁公司</td><td> 430166 </td><td></td></tr><tr><td> Vannas直剪刀</td><td> STORZ眼药水</td><td> E3383小号</td><td></td></tr><tr><td>弯曲的敷料钳锯齿精致</td><td> STORZ眼药水</td><td> E1408 </td><td></td></tr><tr><td> 15度显微刀</td><td>厚朴眼科公司</td><td> 091204 </td><td></td></tr><tr><td>氯胺酮</td><td> Ketaset III </td><td> NADA＃45-290 </td><td></td></tr><tr><td>二甲苯胺噻嗪</td><td>劳埃德实验室</td><td> NADA＃139-236 </td><td></td></tr><tr><td>布比卡因（腔投与局部麻醉药 - 麻佳因） </td><td>阿斯利康公司</td><td> N / A </td><td></td></tr><tr><td>丁丙诺啡</td><td> Sigma Aldrich公司</td><td> B9275 </td><td></td></tr></tbody></table>

subretinal injection of gene therapy vectors and stem cells in the perinatal mouse eye

视力丧失，影响约3.4万人在美国，在未来数年预计将增加。1，基因治疗和干细胞移植已成为主要的治疗手段，治疗视网膜退行性疾病导致的失明。几种形式的自体移植的年龄相关性黄斑变性（AMD），如虹膜色素上皮细胞移植，已经产生了令人鼓舞的结果，为其他形式的基因和干细胞疗法的人体临床试验已经开始。2这些包括RPE65基因置换Leber氏先天性黑蒙和Stargardt病利用人类胚胎干（ES）细胞中的RPE细胞移植治疗的患者。3-4现在有基因治疗的载体，可用于治疗视网膜疾病的患者的干细胞，它是重要的，以验证这些潜在的治疗在动物模型中，前申请ING他们在人类研究中。该鼠标已经成为一个重要的科学模型测试基因治疗的载体和造血干细胞移植在眼的疗效。5-8在这个视频文章中，我们提出了一个技术基因治疗的载体或干细胞分化为视网膜下腔注入老鼠的眼睛，同时最大限度地减少向周围组织的损害。

一个绘图作参考标记的主要结构，示出的鼠标眼玻璃体腔和视网膜下注射的外科手术（箭头， 见图1）的位置显示的箭头。基因治疗的载体，如的LacZ基因的慢病毒载体（ 图2），可以使用这些位置注入。另外，骨髓间充质干细胞，如小鼠胚胎干细胞（ 图3），也可以在这些位点中的老鼠的眼睛被移植。 1微升由巨细胞病毒（CMV）启?...

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Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

这种手术的方法说明了基因治疗的载体和干细胞注射到老鼠的眼睛的视网膜下腔。

基因治疗的载体和干细胞视网膜下注射到老鼠的眼睛

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

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Research

JoVE Journal

Medicine

33.0K 次观看.  Columbia University. 这种手术的方法说明了基因治疗的载体和干细胞注射到老鼠的眼睛的视网膜下腔。

视频: Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a number of gene therapy clinical trials. More recently, a number of additional AAV serotypes have also been isolated, which have been shown to exhibit selective tissue-tropism in various small and large animal models1. Of the 10 most commonly used AAV serotypes, AAV3 is by far the least efficient in transducing cells and tissues in vitro as well as in vivo.
However, in our recently published studies, we have documented that AAV3 vectors transduce human liver cancer - hepatoblastoma (HB) and hepatocellular carcinoma (HCC) - cell lines extremely efficiently because AAV3 utilizes human hepatocyte growth factor receptor as a cellular co-receptor for binding and entry in these cells2,3.
In this article, we describe the steps required to achieve high-efficiency transduction of human liver cancer cells by recombinant AAV3 vectors carrying a reporter gene. The use of recombinant AAV3 vectors carrying a therapeutic gene may eventually lead to the potential gene therapy of liver cancers in humans.

In this article, we describe the identification of the adeno-associated virus serotype 3 (AAV3) as the most efficient vector for targeting human liver cancer cells.

高效率转导肝癌细胞的重组腺相关病毒血清型3载体

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a ...

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 ...

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

在癌症的生产和检测活性氧（ROS）的

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

一种新的手术方法气管内注射生物活性剂在胎鼠模型

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

视网膜脱离模型鼠害由透明质酸钠的视网膜下注射

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

Subretinal Transplantation of MACS Purified Photoreceptor Precursor Cells into the Adult Mouse Retina

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability in industrialized countries. Replacement of degenerated photoreceptors by cell transplantation represents a possible treatment option in future clinical applications. Indeed, recent preclinical studies demonstrated that immature photoreceptors, isolated from the neonatal mouse retina at postnatal day 4, have the potential to integrate into the adult mouse retina following subretinal transplantation. Donor cells generated a mature photoreceptor morphology including inner and outer segments, a round cell body located at the outer nuclear layer, and synaptic terminals in close proximity to endogenous bipolar cells. Indeed, recent reports demonstrated that donor photoreceptors functionally integrate into the neural circuitry of host mice. For a future clinical application of such cell replacement approach, purified suspensions of the cells of choice have to be generated and placed at the correct position for proper integration into the eye. For the enrichment of photoreceptor precursors, sorting should be based on specific cell surface antigens to avoid genetic reporter modification of donor cells. Here we show magnetic-associated cell sorting (MACS) - enrichment of transplantable rod photoreceptor precursors isolated from the neonatal retina of photoreceptor-specific reporter mice based on the cell surface marker CD73. Incubation with anti-CD73 antibodies followed by micro-bead conjugated secondary antibodies allowed the enrichment of rod photoreceptor precursors by MACS to approximately 90%. In comparison to flow cytometry, MACS has the advantage that it can be easier applied to GMP standards and that high amounts of cells can be sorted in relative short time periods. Injection of enriched cell suspensions into the subretinal space of adult wild-type mice resulted in a 3-fold higher integration rate compared to unsorted cell suspensions.

Cell transplantation represents a strategy for the treatment of retinal degeneration characterized by photoreceptor loss. Here we describe a method for enrichment of transplantable photoreceptors and their subretinal grafting into adult mice.

MACS纯化光感受器前体细胞的亚视移植到成年小鼠视网膜中

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability ...

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment epithelium (RPE) cells. RPE provide critical support for photoreceptors and death or dysfunction of RPE cells is characteristic of age-related macular degeneration (AMD), the leading cause of permanent vision loss in people age 55 and older. While no cure for AMD has been identified, implantation of healthy RPE in diseased eyes may prove to be an effective treatment, and large numbers of RPE cells can be readily generated from pluripotent stem cells. Several interesting questions regarding the safety and efficacy of RPE cell delivery can still be examined in animal models, and well-accepted protocols used to inject RPE have been developed. The technique described here has been used by multiple groups in various studies and involves first creating a hole in the eye with a sharp needle. Then a syringe with a blunt needle loaded with cells is inserted through the hole and passed through the vitreous until it gently touches the RPE. Using this injection method, which is relatively simple and requires minimal equipment, we achieve consistent and efficient integration of stem cell-derived RPE cells in between the host RPE that prevents significant amount of photoreceptor degeneration in animal models. While not part of the actual protocol, we also describe how to determine the extent of the trauma induced by the injection, and how to verify that the cells were injected into the subretinal space using in vivo imaging modalities. Finally, the use of this protocol is not limited to RPE cells; it may be used to inject any compound or cell into the subretinal space.

Here we present a community accepted protocol in multimedia format for subretinally injecting a bolus of RPE cells in rats and mice. This approach can be used for determining rescue potentials, safety profiles, and survival capacities of grafted RPE cells upon implantation in animal models of retinal degeneration.

视网膜下进行注射的鼠害提供视网膜色素上皮细胞悬浮

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment ...

Experimental Glaucoma Induced by Ocular Injection of Magnetic Microspheres

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present here a protocol for elevating intraocular pressure (IOP) in the rat, by injecting magnetic microspheres into the anterior chamber of the eye. The use of magnetic particles allows the user to manipulate the beads into the iridocorneal angle, thus providing a very effective blockade of fluid outflow from the trabecular meshwork. This leads to long-lasting IOP rises, and eventually neuronal death in the ganglion cell layer (GCL) as well as optic nerve pathology, as seen in patients with the disease. This method is simple to perform, as it does not require machinery, specialist surgical skills, or many hours of practice to perfect. Furthermore, the pressure elevations are very robust, and reinjection of the magnetic microspheres is not usually required unlike in some other models using plastic beads. Additionally, we believe this method is suitable for adaptation for the mouse eye.

We present a method for inducing elevated intraocular pressure (IOP), by injecting magnetic microspheres into the rat eye, to model glaucoma. This leads to strong pressure rises, and extensive neuronal death. This protocol is easy to perform, does not require repeat injections, and produces stable long-lasting IOP rises.

实验诱发青光眼通过磁性微球注射眼

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

通过重组腺相关病毒载体注射立体的α-突触核蛋白基于大鼠模型为帕金森氏病的发展

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...