这项工作是由美国加州再生医学研究所（RB3-02266）和美国国立卫生研究院（DK54441）的支持。

在开始之前附注： <ol><li>人类胎儿胰腺是从出生缺陷研究实验室，华盛顿大学（西雅图，华盛顿州，美国）谁收获的组织中获得。从捐助者通过该中心获得了组织捐赠的样本，储存和使用的知情同意书。该协议同意声明以书面形式提供，加州大学圣地亚哥分校人类研究保护计划批准整项研究（协议＃081237XT）。 </li><li>它是必不可少的，以保持两者的胎儿胰腺和容器的整个过程中保持在冰上胰腺。解离和消化应尽可能快执行。 </li><li>发送，其中从缓冲存储和胰腺的传输而获得的胎儿胰腺诊所。 （RPMI-1640 +10％正常人血清（NHS），300mM的海藻糖SG，青霉素/链霉素，两性霉素B和）。 </li></ol>人胎儿胰腺1。解剖<ol><li>溶解5毫克在HBSS RT胶原酶XI，得到2.5毫克/毫升的最终浓度。过滤灭菌的溶液用0.22微米注射器过滤器进入无菌（高压灭菌）的闪烁瓶中，并储存于4℃。在组织培养罩，载2的无菌培养皿中，无菌的小坩埚中（如果坩埚是不可用时，一个小烧杯可被取代），无菌剪刀，镊子和。加入2毫升的HBSS一个培养皿洗胰腺。吸出的液体从含有胎儿胰腺，留下大约1毫升管中。 </li><li>除去用钳子胰腺成60毫米的培养皿无HBSS。对于这些实验，是从新鲜的胰腺有妊娠年龄从9到23周胎儿产生IC卡。从胰腺在较早年龄gestionational隔离是困难的，由于胰腺的大小。该协议很可能适用于胎龄超过23周，但收购的pancreata后这段时间，是因为联邦（美国）的限制很难。偶尔，胎儿胰腺到达与脾，脂肪，或附着从原始解剖结缔组织。多余的组织很容易可见的肉眼和应该从起始协议之前胰腺被删除。 </li><li>冲洗胰腺在冷HBSS中（约0.5毫升），在培养皿中的最小体积。 </li><li>将清洗胰腺使用无菌镊子和地点在一个冰桶，小的无菌坩埚。放置在坩埚中的保持器为50ml的离心管中，并使用2对剪刀，大力切片胰腺几分钟。 （一般2-5分钟，但该时间依赖于组织的大小和硬度）。切割技术是非常重要的。在一个固定的位置只移动相反的把手握住剪刀的一侧。剪刀的技巧应该安放在坩埚底部。继续快速剪刀运动打破了胰腺成小件。所述组织的碎片越小越好胶原酶将工作。 </li><li>加入1.5毫升的HBSS +的切碎了胰腺含有胶原酶和地点在水浴摇床设定为37℃，以200rpm的速度长达10分钟的小瓶中。第5分钟时，请勿检查一次更频繁。消化时间依赖于组织的质​​量，少至6分钟就足够了。终点是当颗粒小且均匀。 </li><li>填写小瓶（10-15毫升）与冷的HBSS停止胶原酶活性。放置在冰上，并允许团块沉降10分钟。通常在这一点上，一些细胞死亡发生和DNA被释放到介质中。这可以使媒体的弦“;在这种情况下，添加100μl的DNA酶（10毫克/毫升的库存）到溶液中。 </li><li>后在闪烁瓶中的组织已经结清10分钟，吸出离顶层离开它不到一半略满（7-8毫升）中。将细胞转移到15毫升锥形管，加入5 ml HBSS。离心5分钟，在300×g的。 </li><li>吸出HBSS和悬浮细胞在含有RPMI-1640，GLUTAMAX，10％正常人血清（NHS），10毫摩尔HEPES PH值7.2，青霉素/链霉素，两性霉素B和5毫升媒体。板60毫米的培养皿。研究人员试图生成β细胞，肝细胞生长因子（10毫微克/毫升决赛）添加到媒体。此外，一些团体已经表明，补充培养基与肠促胰岛素激素GLP-1还增加β细胞28。细胞应留在暂停72小时聚集和形成IC卡。 </li><li>经过72小时的悬浮液中，细胞可以被镀在人细胞系HTB9矩阵如前面29所描述的矩阵。无论生长条件，媒体应改为每48小时。 </li></ol>内分泌细胞增殖和成熟标记2。在IC卡中悬浮生长免疫，单层，或者在玻璃盖玻片<ol><li>为了测量细胞的增殖，进行12小时前，PFA固定在悬浮液中或粘附细胞或细胞的BrdU标记。的BrdU试剂1:100稀释并过滤灭菌的RPMI-1640，GLUTAMAX，10％NHS，10毫摩尔HEPES PH值7.0，青霉素/链霉素，两性霉素B和。 </li><li> 对于悬浮细胞：离心5分钟，在300 XG和吸出培养基。加入10 ml PBS中，在300×g离心并吸出洗IC卡，离心5分钟。如果IC卡将要石蜡包埋，请按照任择议定书3.1-3.17 对于贴壁细胞：取出培养基，并如上所述用PBS洗涤细胞。固定细胞20分钟，用4％PFA中于室温，然后用PBS洗两次。在这点上，所述板能缠绕用Parafilm并储存于PBS在4℃下长达1周。 </li><li>孵育的细胞用0.2％的Triton-X 100的PBS在RT下10分钟。用PBS洗涤细胞两次，并应用封闭缓冲液（2％做nkey血清，2％牛血清白蛋白，50mM的甘氨酸1小时在PBS中稀释）。用工作缓冲液洗涤细胞（封闭缓冲液稀释后1:10）。稀释的抗体在工作缓存器。对于生长在玻璃盖玻片上的细胞，稀释的抗体在60微升的总体积。对于细胞的6孔培养皿，稀释抗体在600微升总体积。染色多个表位，抗体的鸡尾酒可能被应用。作为对照，使用IgG或预免疫血清代替特定的抗体。控制样品必须孵育来自相同物种的对照IgG作为正在使用的一抗。 </li><li>孵育一抗为1.5小时，在室温或O / N在4°C。吸出第一抗体，并与工作缓冲液洗3次在RT。 </li><li>稀释的第二抗体在1:200罗丹明和FITC标记的抗体，1:500 - 1:1000的Alexa的共轭抗体。要注意的是所有的次级抗体是光敏感是很重要的。盖上样品铝箔，以防止信号损失。孵育1小时，在室温。 可选：要可视化的细胞核，DAPI可以在1:500二级孵化过程中添加。这是用于蛋白质表达中的总的细胞群体的定量是有用的。 </li><li>吸出的第二抗体，并与工作缓冲液洗3次在RT。对于生长在盖玻片上的细胞，轻轻提起盖玻片用钳子将其浸在缓冲;通过浸入在PBS中洗涤。在一个6孔板中生长的细胞，加入PBS洗并抽吸。在这一点上，他们准备在倒置显微镜下检查。 </li><li>触摸盖玻片的边缘上轻轻一的Kimwipe摆脱多余的PBS。添加安装凝胶载玻片上滴和反转盖玻片上的幻灯片。通过抽吸去除多余的安装胶。点击盖玻片轻轻地用200μl的枪头，赶走气泡的背面。 </li><li>在室温下干燥约20分钟，或O / N在4℃，并根据佛罗里达州考察uorescent或共聚焦显微镜。 </li></ol>从石蜡包埋的ICC蛋白3（可选）免疫荧光染色<ol><li>准备3％琼脂糖溶液，放冷至55-60℃。 </li><li>转移的IC卡中温育悬浮到15ml锥形管中，并离心分离机以1500×g离心5分钟，在室温下进行。 </li><li>从细胞中吸出培养基，用500μl的4％PFA中于室温10分钟，固定。不要混合颗粒，除非颗粒是非常大的。 </li><li>用750微升PBS洗两次沉淀。像4％PFA，这种废物被认为是危险的，应该根据自己的机构的危险废物规范处置。 </li><li>轻轻轻拂细胞沉淀松动，并添加150-200微升的琼脂糖下来的的15毫升锥形管的壁。 </li><li>轻轻弹管混匀，但不要形成气泡。 </li><li>允许凝固在4℃下5-10分钟。 </li><li>使用21号针头撞出颗粒并将其放置在一个新的15毫升锥形管。 </li><li>石蜡包埋和切片的制备上的幻灯片可以在现场用切片机或你的核心显微镜设备来完成。 </li><li> Deparaffinize组织，水合物，并用水迅速洗。 </li><li>添加0.2M的甘氨酸在室温30分钟以淬灭残留的醛。 </li><li>每个2分钟，用PBS洗两次的幻灯片。 </li><li>用于抗原修复，带来的10mM柠檬酸缓冲液（pH 6.0）中煮沸过的热板。沉浸幻灯片在缓冲区中，等待它回来烧开，然后等待15分钟。 </li><li>从电炉中取出烧杯中。等待大约40分钟的幻灯片在缓冲区中冷却。 </li><li>与H 2 O洗两次的幻灯片，每次5分钟，用PBS洗两次的幻灯片，每次5分钟，以1小时的PBS 2％正常驴血清阻断。 </li><li>孵育一抗，稀释至含0.2％的Triton X-100浓度正确工作缓冲区。通常，初级抗体孵育过夜，如果染色为转录因子和1.5小时，如果染色为激素，细胞增殖的标记物，和/或分化。 </li><li>遵循上述步骤2.3-2.9。 </li></ol>

<ol>
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I., Ricordi, C., Hayek, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotinamide+is+a+potent+inducer+of+endocrine+differentiation+in+cultured+human+fetal+pancreatic+cells.">Nicotinamide is a potent inducer of endocrine differentiation in cultured human fetal pancreatic cells.</a> J Clin Invest. 92, 1459-1466 (1993).</li><li>Xie, 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=Dynamic+chromatin+remodeling+mediated+by+polycomb+proteins+orchestrates+pancreatic+differentiation+of+human+embryonic+stem+cells.">Dynamic chromatin remodeling mediated by polycomb proteins orchestrates pancreatic differentiation of human embryonic stem cells.</a> Cell Stem Cell. 12, 224-237 (2013).</li><li>Hardikar, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+maturation+of+fetal+porcine+beta-cells+by+glucagon-like+peptide+1+and+cholecystokinin.">Functional maturation of fetal porcine beta-cells by glucagon-like peptide 1 and cholecystokinin.</a> Endocrinology. 143, 3505-3514 (2002).</li><li>Beattie, G. M., Cirulli, V., Lopez, A. D., Hayek, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+expansion+of+human+pancreatic+endocrine+cells.">Ex vivo expansion of human pancreatic endocrine cells.</a> J Clin Endocrinol Metab. 82, 1852-1856 (1997).</li><li>Beattie, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trehalose:+a+cryoprotectant+that+enhances+recovery+and+preserves+function+of+human+pancreatic+islets+after+long-term+storage.">Trehalose: a cryoprotectant that enhances recovery and preserves function of human pancreatic islets after long-term storage.</a> Diabetes. 46, 519-523 (1997).</li><li>Hayek, A., Beattie, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+transplantation+of+human+fetal+and+adult+pancreatic+islets.">Experimental transplantation of human fetal and adult pancreatic islets.</a> J Clin Endocrinol Metab. 82, 2471-2475 (1997).</li><li>Otonkoski, T., Beattie, G. M., Lopez, A. D., Hayek, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+hepatocyte+growth+factor/scatter+factor+to+increase+transplantable+human+fetal+islet+cell+mass.+Transplant.">Use of hepatocyte growth factor/scatter factor to increase transplantable human fetal islet cell mass. Transplant.</a> Proc. 26, 33-34 (1994).</li><li>Beattie, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+approach+to+increase+human+islet+cell+mass+while+preserving+beta-cell+function.">A novel approach to increase human islet cell mass while preserving beta-cell function.</a> Diabetes. 51, 3435-3439 (2002).</li></ol>

作者宣称，他们有没有竞争的财务权益。

这里介绍的方法，使人们能够从胎儿胰腺和随后图像进行细胞增殖和胰腺内胚层标志物生成的ICC。人胎儿胰脏分解的过程需要约90分钟，接着进行72小时的ICC形成时期，这种方法是从协议实质上不同，从小鼠分离胰岛β细胞祖细胞。这里介绍的协议提供了一个可重复的方法，使研究人员探索人类胎儿胰腺发育。两种不同类型的纵向研究可以被执行。首先，从不同胎龄产生功能性胰岛细胞类型（导管细胞，外分泌细胞，和激素阳性细胞）的电位可移植后进行评估。其次，从单一的胎龄IC卡可在培养基中培养，与选定的药剂处理和ICC文化移植过程中不同时间点，探索差异在细胞的命运。与目前正在针对人类胚胎干细胞分化为胰岛素分泌细胞的巨大努力，与胰岛素分泌反应的生理刺激相关的问题再次被恢复。人类IC卡提供了一个独特的模型系统来研究胎儿胰腺细胞增殖和β细胞发育这一关键方面。 正如任何协议从组织分离细胞，细节是成功的关键。许多参数是不幸执行实验的实验室人员的控制范围之外。本实验室遇到的两个问题包括原料质量差和交付非胰腺组织。健康的胎儿胰腺组织是坚定的剪纸。如果组织削减太容易，这是一个迹象，表明胰腺酶开始消化胰腺本身。从这个不幸的是没有恢复和准备，最好取消。偶尔，在一个经验丰富的技术人员取出胰脏会混淆肠道的各个部分的胰腺。这些样本将有超过胰腺更加弹性和管腔显着将出席。同样，这些样本应该被丢弃。 当上述协议所描述的后面，还有极少出现的扔隔离偏离轨道的任何问题。有几点要记住，以确保顺利隔离包括，用锋利的剪刀进行精确切割胰腺并确保不留下任何组织样本过大消化。如果裁员是不是足够小，那么胶原酶不奏效，很少的ICC形成。相反，使用一批新的胶原酶的时候，开始用类似的水平，如果IU（国际单位）的消化与先前使用。然而，减少温育时间，以确保通过消化不发生。此故障排除技术，保证了IU的标签不显著批与BA有所不同TCH。 在拍摄的角度来看，这种技术对人类胎儿IC卡的隔离是比较标准的领域。大多数实验室证实了我们的调查结果，除了肝细胞生长因子的媒体可以帮助细胞存活和增殖33。总之，我们已经开发出一种方法来分离人胎儿的ICC新鲜胰腺妊娠年龄从9到23周。该细胞可生长为单层或悬浮液中进行实验，以可视化的胰腺内分泌转录因子和人胰岛素。

一个主要限制在1型糖尿病患者的细胞为基础的胰岛素替代疗法可用于人体移植胰岛的缺乏。调节细胞增殖人体胰腺前体细胞，并分化成符合的胰岛素缺乏状态下的代谢需求产生胰岛素的细胞的能力仍然是一个关键的构建块一个基于细胞的疗法来治疗1型糖尿病。 直到人类胚胎干细胞衍生的产生胰岛素的细胞，人胎儿胰腺内分泌细胞或其前体的出现被认为是潜在的细胞用于临床移植的来源。虽然科学和监管环境在过去几年发生了变化，仍然非常需要了解如何在人类胎儿胰腺发展。许多人现在查看的治疗利用人体胚胎细胞的可能性不大，但如果有效，安全的方法来扩大细胞建立，治疗使用这些细胞可能AGAIN进行探讨。这仍然是一个主要障碍是， 在人类胎儿胰腺细胞聚集体（IC卡）的体外转化为葡萄糖敏感，胰岛素分泌的内分泌细胞是目前一个低效的过程。尽管还有许多工作过近30年来阐明和界定所需的内分泌胰腺的发育转录因子的表达谱，但留在我们关于如何转录因子时空表达调控及与细胞功能的知识差距。 直到人类胚胎干细胞衍生的产生胰岛素的细胞，人胎儿胰腺内分泌细胞或其前体的出现被认为是潜在的细胞用于临床移植的来源。虽然科学和监管环境在过去几年发生了变化，仍然非常需要了解如何在人类胎儿胰腺发展。许多人现在查看的治疗利用人体胚胎细胞的可能性不大，但如果有效和安全的方法来扩展建立的细胞，治疗使用这些细胞可以再次进行探讨。这仍然是一个主要障碍是， 在人类胎儿胰腺细胞聚集体（IC卡）的体外转化为葡萄糖敏感，胰岛素分泌的内分泌细胞是目前一个低效的过程。尽管还有许多工作过近30年来阐明和界定所需的内分泌胰腺的发育转录因子的表达谱，但留在我们关于如何转录因子时空表达调控及与细胞功能的知识差距。 最近，干细胞领域已采用胰岛发育过程中对时间的转录因子表达积累的知识来驱动生产细胞表达成熟的内分泌细胞的标志物。虽然从人类胚胎干细胞和诱导多能干细胞（IPSC）的胰岛素生产细胞的起源已取得实质性和在过去的几年中显著进步，最有效的协议需要两个不同的分化阶段：1）早期在体外分化产生细胞表达胰前体的转录因子，其次为2） 在体内成熟移植后-即所谓的“黑盒“期。前进，前进理解在体内的生物学基本胰岛成熟，无论细胞来源，必须在生化水平被理解。与IC卡和人类胚胎干细胞所获得的结果之间的相似性表明，许多调节人胰腺癌细胞前体细胞的过渡到成熟，葡萄糖响应性，胰岛素分泌细胞的体外关键生化过程仍不清楚。这种理解的核心部分将开​​发新的方法来从胰腺祖细胞中获得功能的内分泌细胞群和人类胚胎干细胞不仅需要生化APPRoaches该澄清的成熟的事件，但方法的变化来分析。 为什么我们相信成像人类胎儿胰腺细胞的识别变化胰岛成熟的一个重要方面？答案在于部分在历史的追求，产生胰岛素的细胞。这两个模型和组织培养系统，胰腺前体和小岛已使研究人员能够探索动物胰岛细胞和人类胰岛体外成熟之间存在的显著差异。一个限制对人类胎儿胰腺发育的探索是，胎龄，可以合法使用只异类细胞聚集导致。虽然分离的人胎儿细胞类似胰岛，染色体外培养从胎龄9-23周后显示小于15％的包含标记为内分泌细胞，与未染色胰岛激素大部分细胞。然而，移植后的体内成熟，绝大多数细胞表达内分泌标记6,25。从这些研究的结果正在今天回荡着人类胚胎干细胞分化的协议是唯一能够产生单一激素阳性内分泌细胞的适度种群在体外分化之后， 在体外方法以增强激素阳性细胞群可能会提供深入了解体内胰岛成熟。此外，了解分子事件推动人类胎儿胰腺发育很可能会提高努力从人类胚胎干细胞衍生的胰岛素制造细胞，并进一步划定调节β细胞的再生和胰腺细胞转分化的机制。 人胎胰腺细胞和人类胚胎干细胞的成熟之间的相似性可以扩展到细胞的功能。像鼠类细胞，人类胎儿细胞和分化的人类胚胎干细胞不能分泌胰岛素响应于葡萄糖后在体外 26,27胰岛新生物。然而，移植到裸鼠和成熟，人类胰岛样集群和从人类胚胎干细胞表现出一种内分泌源性表型胰岛素产生细胞时。移植后三个月，被移植的细胞无论从人口的几乎90％的胰岛素阳性，并正常运行，就通过C-肽17,26的释放来确定。这些结果表明，移植的规定，促进胰岛成熟的背景和身份不明的线索。优化的成像协议，如这里描述的，对于人胎儿胰腺细胞会在搜索中鉴定调节，加速成熟因子帮助1。基本生化探索人类胎儿胰腺细胞和人类胚胎干细胞在这个发展阶段的差异化迈向内分泌系是衡量成熟的效率至关重要。 下面的协议，在FIGUR概述E 1，提供了我们目前的方法对于人胎儿胰腺细胞的分离，称为IC卡从这些细胞的整个胎儿胰腺和成像。此协议需要细胞从组织中的初始制剂，其随后可以生长为单层或悬浮液中。描述细胞的内分泌细胞增殖和成熟常用的影像学标志物的制备。 生成和IC卡的成像多种化学改性剂的存在与否提供了一个快速的方法，与移植模型相比，以帮助确定培养条件和加速人类胎儿胰腺细胞发育成完整功能的外分泌，乳腺导管，或化合物激素的胰脏细胞。

<table border="0" cellpadding="0" cellspacing="0" width="1000">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:349px;">Trehalose SG</td>
			<td style="width:219px;">Hayashibara</td>
			<td style="width:115px;">Trehalose SG</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase XI&#160;</td>
			<td>Sigma</td>
			<td>C-9407</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hank&#8217;s Balanced Salt Solution (HBSS)</td>
			<td>Life Technologies</td>
			<td>24020-117</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI 1640 with glutamate</td>
			<td>Life Technologies</td>
			<td>11879020</td>
			<td>no glucose or HEPES</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human AB Sera, Male Donors</td>
			<td>Omega Scientific</td>
			<td>HS-30</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fungizone</td>
			<td>Life Technologies</td>
			<td>15290018</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gentamicin Solution 50 mg/ml</td>
			<td>Invitrogen</td>
			<td>15750060</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1M Hepes, pH 7.0</td>
			<td>Life Technologies</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin - Streptomycin 100X Solution</td>
			<td>Life Technologies</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glutamax</td>
			<td>Life Technologies</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hepatocyte Growth Factor (HGF)</td>
			<td>Peprotech</td>
			<td>100-39</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GLP-1</td>
			<td>Peprotech</td>
			<td>130-08</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum</td>
			<td>Invitrogen</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dnase</td>
			<td>Sigma</td>
			<td>DN25</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile Petri Dishes, 60 x 15 mm; Stackable, venting ribs</td>
			<td>Spectrum Laboratory Products, Inc.</td>
			<td>D210-13</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS</td>
			<td>Gibco</td>
			<td>14190</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">16% PFA</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triton X-100</td>
			<td>Sigma</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Donkey Serum&#160;</td>
			<td>Jackson ImmunoResearch</td>
			<td>017-000-121</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BrdU</td>
			<td>Life Technologies</td>
			<td>00-0103</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-insulin (mouse monclonal; 1:1000)</td>
			<td>Sigma</td>
			<td>I2018</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-glucagon (mouse monoclonal; 1:2000)</td>
			<td>Sigma</td>
			<td>G2654</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PDX1 (mouse monoclonal)</td>
			<td>Novus Biologicals</td>
			<td>NBP1-47910</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PDX1 (goat polyclonal)</td>
			<td>AbCam</td>
			<td>47383</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PDX1 (rabbit polyclonal)</td>
			<td>AbCam</td>
			<td>47267</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AlexaFluor 546 (rabbit)</td>
			<td>Invitrogen</td>
			<td>A11010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AlexaFluor 546 (mouse)</td>
			<td>Invitrogen</td>
			<td>A11003</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AlexaFluor 488 (rabbit)</td>
			<td>Invitrogen</td>
			<td>A11008</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AlexaFluor 488 (mouse)</td>
			<td>Invitrogen</td>
			<td>A11001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ki67</td>
			<td>Lab Vision Neomarkers</td>
			<td>RM 9016-50</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pan-CK (mouse monoclonal; 1:100)</td>
			<td>Immunotech</td>
			<td>2128</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CK19</td>
			<td>AbD Serotech</td>
			<td>MCA 2145</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAPI</td>
			<td>Cell Signaling</td>
			<td>4083</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HTB9 cell line</td>
			<td>ATCC</td>
			<td>5637</td>
			<td>see Beattie 1997 reference to generate matrix</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agarose</td>
			<td>Sigma</td>
			<td>A-6013</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation, culture, and imaging of human fetal pancreatic cell clusters

近30年来，科学家们已经证明了在裸鼠 ​​肾囊移植人胎IC卡已发展成为功能的内分泌细胞，证明了循环人C-肽下列葡萄糖刺激1-9显著增加。然而， 在体外，从人胎IC卡胰岛素制造细胞的起源是低10;结果让人联想起人类胚胎干细胞（胚胎干细胞）最近进行的实验中，细胞大有希望作为一个潜在的治疗治疗1型糖尿病的可再生能源。 IC卡一样，部分分化的人类胚胎干细胞的移植产生葡萄糖响应，产生胰岛素的细胞，但在从人类胚胎干细胞的胰岛素分泌细胞的体外起源要少得多健壮11-17。对影响内分泌前体细胞的生长和分化的因素一个完整的理解可能会需要来自国际商会和他产生的数据资深大律师。虽然许多协议存在，从人类胚胎干细胞产生的胰岛素生产细胞在体外 11-22，为国际商会10,23,24少得多的存在。该差异的一部分可能来自与人胎胰腺工作的难度。为此，我们继续建立在现有的方法分离胎儿胰岛细胞从人类胰腺与孕龄介乎12至23周，成长的细胞作为单层或悬浮和​​图像细胞增殖，胰腺癌标志物和人体激素包括胰高血糖素和C-肽。 IC卡通过在裸鼠 ​​类似于由新鲜组织6移植获得的C-肽水平的肾囊移植后的C-肽释放导致如下所述的协议产生。虽然这里给出的例子讨论在胰腺内胚层细胞增殖和β细胞发生，该协议可以用于研究胰腺发育的其他方面包括外分泌，导管和其他激素生成细胞。

精心准备的胎儿IC卡的是该协议的成功是至关重要的。如何在细胞通常看定义期间电镀后代表图片如图2。纯化后，刚镀细胞聚集体出现包含少数细胞（图2A）小，疏清集群。在第一个24小时（图2B）后，许多细胞簇的变大，但众多的小簇仍然存在。 48小时，该集群已显著扩大，但仍不对称（图2C）。在72小时后，将IC卡应该是明确的，相对统一的大小和形状（图2D）。它是在这一点上，这些细胞可以被任一铺于基质，如HTB-9，放置24小时前，免疫荧光或允许在不存在或化学品或药物的存在可能改变基因表达的生长另外72小时所需的胰腺内分泌细胞克eneration。 图3突出显示了一些常用的抗体来评估或者ICC增殖或分化对胰腺内胚层。在图3A中 ，IC卡被镀上HTB-9，生长4天，并染为PDX1，胰腺发育的关键指标。虽然在体外染色IC卡是在我们的实验室常规进行，更多的时候，人胎IC卡是根据裸鼠肾囊移植，并使其增殖和分化的体内 6,9,30-32。在移植后的指定时间，将小鼠处死并取出含有移植的ICC肾脏，固定在4％多聚甲醛固定，并包埋于石蜡中。无论是在现场用切片机或核心显微镜设备产生连续5微米的部分。抗原表位的揭露和石蜡包埋组织进行免疫荧光蛋白染色任择议定书3.10-3描述。17在图3B中，移植的IC卡进行染色上皮细胞标记物泛细胞角蛋白（PanCK;绿色）及与Ki67（红色）来评估细胞增殖。在另一个例子中，人胰岛素（绿色）和胰高血糖素（红色）下的裸鼠（图3C）的肾囊移植和成熟后进行检测。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/50796/50796fig1highres.jpg" width="500" /> 图1。流程图人类胎儿的ICC制备和染色的免疫荧光。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/50796/50796fig2highres.jpg" width="500" /> 图2。代表胎儿的ICC准备为0，电镀后24，48，或72小时（一）IC卡在悬浮液中进行成像电镀后立即，（B）24小时后镀，电镀，或（D）电镀后72小时后（℃）48小时。比例尺为AC，10倍放大倍率= 300微米，比例尺为D，20倍= 100微米。 <a href="https://www.jove.com/files/ftp_upload/50796/50796fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/50796/50796fig3highres.jpg" /> 。图3免疫荧光镀金IC卡按常规，IC卡被用于成像（一）胰腺内胚层标志物PDX1（绿色），（B）内分泌细胞（泛长江，绿色）和增殖（Ki67的，红色），（C）胰岛素（绿色）和胰高血糖素（红色）。比例尺为AC，40X放大倍率= 20微米。

Watch this Scientific Journal Video about Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters at JoVE.com

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

一个协议隔离开来，从人胎儿胰腺细胞衍生的文化，形象胰岛细胞团（IC卡）中说明。该方法的详细信息需要从组织，文化为单层或悬浮液中的聚集和图像增殖的标记和胰腺细胞命运的决定产生IC卡的步骤。

分离，培养和成像胎儿胰腺细胞集群

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

isolation-culture-and-imaging-of-human-fetal-pancreatic-cell-clusters

Research

JoVE Journal

Medicine

14.0K 次观看.  University of California, San Diego. 一个协议隔离开来，从人胎儿胰腺细胞衍生的文化，形象胰岛细胞团（IC卡）中说明。该方法的详细信息需要从组织，文化为单层或悬浮液中的聚集和图像增殖的标记和胰腺细胞命运的决定产生IC卡的步骤。 

视频: Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. 
Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region.
The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&amp;E and IHC stains. 
The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&amp;E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.

人类胰岛染色协议

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

科研

医学

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

婴幼儿的脑代谢和血流动力学的非侵入式光学测量

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

Experimental examination of normal human mammary epithelial cell (HMEC) behavior, and how normal cells acquire abnormal properties, can be facilitated by in vitro culture systems that more accurately model in vivo biology. The use of human derived material for studying cellular differentiation, aging, senescence, and immortalization is particularly advantageous given the many significant molecular differences in these properties between human and commonly utilized rodent cells1-2. Mammary cells present a convenient model system because large quantities of normal and abnormal tissues are available due to the frequency of reduction mammoplasty and mastectomy surgeries.
The mammary gland consists of a complex admixture of many distinct cell types, e.g., epithelial, adipose, mesenchymal, endothelial. The epithelial cells are responsible for the differentiated mammary function of lactation, and are also the origin of the vast majority of human breast cancers. We have developed methods to process mammary gland surgical discard tissues into pure epithelial components as well as mesenchymal cells3. The processed material can be stored frozen indefinitely, or initiated into primary culture. Surgical discard material is transported to the laboratory and manually dissected to enrich for epithelial containing tissue. Subsequent digestion of the dissected tissue using collagenase and hyaluronidase strips stromal material from the epithelia at the basement membrane. The resulting small pieces of the epithelial tree (organoids) can be separated from the digested stroma by sequential filtration on membranes of fixed pore size. Depending upon pore size, fractions can be obtained consisting of larger ductal/alveolar pieces, smaller alveolar clusters, or stromal cells. We have observed superior growth when cultures are initiated as organoids rather than as dissociated single cells. Placement of organoids in culture using low-stress inducing media supports long-term growth of normal HMEC with markers of multiple lineage types (myoepithelial, luminal, progenitor)4-5. Sufficient numbers of cells can be obtained from one individual's tissue to allow extensive experimental examination using standardized cell batches, as well as interrogation using high throughput modalities.
Cultured HMEC have been employed in a wide variety of studies examining the normal processes governing growth, differentiation, aging, and senescence, and how these normal processes are altered during immortal and malignant transformation4-15,16. The effects of growth in the presence of extracellular matrix material, other cell types, and/or 3D culture can be compared with growth on plastic5,15. Cultured HMEC, starting with normal cells, provide an experimentally tractable system to examine factors that may propel or prevent human aging and carcinogenesis.

A method to process human mammary surgical discard material is described. Processed tissue, in the form of organoids, can be stored frozen indefinitely or placed in culture for long-term growth. This method enables experimental examination of normal human epithelial cell biology, and the effects of exogenous perturbations.

处理人减少乳房整形和乳房切除术的组织细胞培养

Experimental  examination of normal human mammary epithelial cell (HMEC) behavior, and how  normal cells acquire abnormal properties, can be facilitated ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

来自患者的细胞培养和分离的CD133<sup&gt; +</sup&gt;假定的肿瘤干细胞的恶性黑色素瘤

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

从手术标本的人类胰岛细胞激光显微切割的改进方法

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

分离，冻存和文化的人羊膜上皮细胞的临床应用

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

对无瘢痕修复鼠标胎儿皮肤模型

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

瘦素受体<sup&gt;分贝</sup&gt; 2型糖尿病小鼠模型：胰岛分离和活细胞双光子成像完好胰岛

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

的肿瘤特异性内皮细胞的分离与培养扩展

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized stromal cells and immune cells, are responsible for the secretion of hormonal and inflammatory factors which are critical for successful blastocyst implantation, placental development and play a role in the initiation of labor at term and preterm. Many pregnancy complications can arise from perturbations of a fine balance of different cell populations comprising decidua. Alterations in the proportion of specific decidual cell types may disrupt these crucial processes and increase the risk of developing serious complications of pregnancy, such as embryo implantation failure, intrauterine growth restriction, preeclampsia and preterm labor. The protocol outlined here demonstrates a cost and time effective method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae. By combining enzymatic digestion and gentle mechanical disruption of the decidual tissue, a high yield of decidual cells was obtained with virtually no chorion contamination. Importantly, isolated decidual cells were characterized (stromal cells (55-60%), leukocytes (35%), epithelial (1%) or trophoblast (0.01%) cells) and maintained high viability (80%) which was confirmed by multicolor imaging flow cytometry assay. This protocol is specific to the decidua parietalis and can be adapted to first and second trimester placentae. Once isolated, decidual cells can be used for a multitude of experimental applications aiming to understand the role of different decidual cell sub-populations in pregnancy complications.

This protocol demonstrates a method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae which can be used for a variety of applications (i.e. immunocytochemistry, flow cytometry, etc.) aiming to study the role of different cell populations in pregnancy complications.

足月胎盘胎膜中原发性人蜕细胞的分离

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...