我们希望大学由Arieh已Shalhav博士率领芝加哥科泌尿外科，泌尿研究嘉莉博士RINKER谢弗主任的支持表示感谢。我们还想大学芝加哥综合癌症中心（UCCCC）LE BEAU米歇尔博士领导的支持表示感谢。我们还与，感谢专家技术援助的人体组织资源中心的核心设施马克林根博士的带领下，和莱斯利·马丁和玛丽·乔·菲克特援助。我们也感谢李特丽免疫组化核心设备运行。这项工作是由芝加哥外科部泌尿外科，该​​科大学，美国癌症协会的研究资助机构（ACS-IRG IRG-58-004）;癌症中心的支持格兰特（P30 CA14599）;布林森基金会;阿尔文鲍姆家庭基金;大学芝加哥癌症研究基金会的董事会; S.克雷格尔由霍华德休斯医学研究所的支持:到毕业的医学研究员髋部（56006772）和癌症生物学训练津贴（T32-CA09594）。最后，我们要感谢罗伯特·克拉克，和弥敦道Stadick博士VenkateshKrishnan，都为他们的批判性评价的稿件。

这项研究进行了严格按照美国国立卫生研究院的实验室动物护理和使用指南的建议。该协议被批准大学芝加哥机构动物护理和使用委员会（IACUC，协议号码72066和72231）。所有的手术麻醉下进行，所有的努力都是为了最大限度地减少痛苦。收购WiCell（麦迪逊，威斯康星州）和培养人类胚胎干细胞系WA01（H1; NIH注册＃0043）使用独立的馈线协议使用mTeSR1媒体（干细胞技术;温哥华，不列颠哥伦比亚省）。胚胎干细胞被用来内10通道解冻。 1。从小鼠或大鼠胚胎的泌尿生殖窦分离<ol><li>安乐死定时孕鼠（17.5天）或大鼠吸入CO 2（18.5天）5分钟。也证实了在大鼠的生命体征停止死亡。然后打破它的脖子人道。在R在安乐死在这一点上。喷70％乙醇消毒腹部皮肤和侧面。切腹部的皮肤和肌肉层，然后切子宫，分离大鼠胚胎的羊膜囊，剪断脐带。胚胎转移到一个50毫升的Falcon管中含有冰冷的Hank氏平衡盐溶液（HBSS）中，并保持在冰上。胎儿安乐死斩首用手术剪刀。 </li><li>将胚胎在100毫米的培养皿。平分胚胎用剪刀在肚脐的水平（ 图1A）。取出胃和小肠。显示女性胚胎卵巢和睾丸在男性胚胎（ 图1B和1C）。 （卵巢位于尾侧磁极的肾脏，睾丸是大致水平的膀胱）。在这个发展阶段，UGSM隔离的主机的存在能够诱导前列腺上皮血统的男性和女性胚胎然而，这个发展的时间点后的睾丸激素。4，前列腺芽开始形成UGSM，和纯UGSM隔 ​​离是非常具有挑战性的。 </li><li>解剖显微镜下，在中间线与维纳斯剪刀露出腹部膀胱后壁切开腹壁。免费上生殖道的附件（雄性胚胎，切断输尿管，沃尔夫管和苗勒氏管;女性胚胎，切断输尿管和苗勒氏管）。从脐带释放膀胱。切耻骨直言分开杜蒙精尖镊子泌尿生殖窦前壁。最后，从直肠泌尿生殖窦后壁分开。在其下端（从尿道）剪切与膀胱的泌尿生殖窦和冰冷的HBSS中存储的泌尿生殖窦整块（膀胱）。 </li></ol> 2。泌尿生殖窦间质细胞的分离<ol><li&gt;解剖显微镜下取出膀胱泌尿生殖窦（ 图1D）。 </li><li>转移泌尿生殖窦含1％胰蛋白酶，钙（Ca 2 +的）和镁（Mg 2 +）的HBSS猎鹰管。管放置在冰箱中在4℃下75分钟。 </li><li>取出胰蛋白酶和10％胎儿的鲍文血清（FBS）的DMEM/F12培养基中和胰酶消化。 </li><li>小心地挑逗粘间充质套管针或的杜蒙细尖镊子（保持完好的上皮管上皮细胞间质污染的机会减少，这可能会导致啮齿动物的腺体形成，随着干细胞衍生的人类上皮管腺上皮细胞）（ 图1D）。 </li><li>转移到一个新的猎鹰管含有DMEM/F12培养基+ UGSM 10％FBS + 1％青霉素/链霉素（PEN /链球菌，0.5毫克/毫升）+ 1％非必需氨基酸（NEAA）加1nm的R1881。排位一点管的培养箱中在37℃用5％CO 2下过夜。 </li><li>组织以200×g离心，弃去上清液。洗净用磷酸盐缓冲液（PBS）（请勿打扰组织沉淀）。 </li><li>添加0.2％胶原酶的DMEM/F12重悬组织。在37°C孵育1小时，温和的摇摆。 </li><li>大力涡消化组织三次，直到它变成均匀的单细胞混合物中，并加DMEM/F12培养基+ 10％FBS的1％P / S + 1％NEAA + 1nm的R1881中的胶原酶。 </li><li>在200 XG离心，然后洗重新悬浮细胞在HBSS UGSM，重复3次充分洗净UGSM的。最后暂停的DMEM/F12间质细胞计数使用血球细胞或细胞计数装置。 </li><li>游离培养胚胎干细胞使用馈线协议（mTeSR1媒体干细胞技术，不列颠哥伦比亚省温哥华）使用的Accutase消化（Millipore公司;比尔里卡，MA）。洗净的胚胎干细胞DMEM/F12媒体在温暖他们的成长在日志记录阶段，再加入温暖的1X的Accutase解决方案，并在37℃孵育15-20分钟，直到单细胞均匀脱离殖民地。新增等量，1X定义的胰蛋白酶抑制剂（Invitrogen公司，大岛，NY）中的Accutase轻轻吸取向上和向下混合，打破了细胞团块。移液管细胞以200×g，3分钟，重新悬浮细胞沉淀中的DMEM/F12培养基到离心管中，离心。离心管3分钟在200 XG再次和去除上清，然后重新暂停在冷的HBSS或DMEM/F12所需的音量。 </li><li>胚胎干细胞与胚胎干/间充质细胞的1:2.5的比例添加。3，这将是一个蜂窝1E 5 UGSM的4E 4每10毫升注射（单个大鼠UGS通常约2E 6间充质细胞的产率）的人类胚胎干细胞的细胞组合物。离心200×g离心5分钟，然后弃去上清液。重新暂停与75％基质胶（BD Biosciences公司，加利福尼亚州圣何塞，混有25％冷的HBSS更容易处理），然后将子肾囊注射管在冰。 </li></ol> 3。移植肾包膜底下的组织重组<ol><li>准备一个无菌手术区。 </li><li>麻醉裸鼠鼠标一个男性氯胺酮/甲苯​​噻嗪IP，使用新鲜的组合，包含1:1:8的比例氯胺酮:甲苯噻嗪:生理盐水。给药将是100毫克/千克氯胺酮和2.5毫克/公斤甲苯噻嗪。一个脚趾捏反应迟钝的动物应该是完全由20-30分钟。 </li><li>手术准备剃须手术部位（如果不使用裸鼠），鼠标，清洁三个交替湿巾70％的酒精，然后用优碘溶液和应用手术悬垂。 </li><li>将眼部水分的药膏（Altalube眼膏）鼠标的眼睛，在麻醉过程中，以防止干燥。 </li><li>在手术过程中，小鼠的呼吸速率与核心体温监控。呼吸率升ULD稳定，保持在40-90次/分钟。芯体的温度进行监控，观察粘膜和颜色的粉红色的脚底。 </li><li>一个1.0厘米长的纵向皮肤切口的背。继续在0.5厘米纵切口切开腹壁。 </li><li>轻轻挤压腹部和肾肾脏保持表面湿润，用无菌生理盐水滴。 </li><li>轻轻穿刺肾包膜使用一个空的27号注射器针头。一个非常浅刺就足够了。这将是您插入您的钝针注射的细胞混合物。 </li><li>用10微升的细胞混合物中填写的28号钝的注射器针头。慢慢插入穿刺部位和穿透肾实质面积将达肾包膜下方。注入10μl的细胞混合物以形成丸剂对肾脏肾小囊的下面。提款针。 </li><li>肾脏阿卜杜接线腔。固定用4-0尼龙缝合腹膜肌肉层。 </li><li>关闭的皮肤，无论是缝合或主食。 </li><li>清洁血液从皮肤，用无菌生理盐水。 </li></ol> 4。手术后的镇痛和监测<ol><li>丁丙诺啡（0.1毫克/千克，每12小时）或酮洛芬（3-5毫克/公斤，盐水每隔12小时）注入动物管理手术后的疼痛，1毫升37°C生理盐水IP动物保持温暖并防止脱水。 </li><li>将在一个干净的笼子里的动物，并把它放在一个温和的加热垫。 </li><li>发送的动物，动物房，一旦他们恢复知觉，并正在内笼。 </li><li>意想不到的疾病和感染迹象随后三天，每天观察动物。将小鼠观察在笼子里，包括存取的食物和水后1天可操作的活动监测。如果老鼠显示活动较少，酮洛芬是腹腔广告再次伺候，每天一次。 </li><li>去除皮肤钉或缝线手术后2周。 </li><li>异种移植组织可以尽早手术后8周收获。使用小动物超声（ 图2A），可以是固定的组织切片，染色用免疫组化实验（ 图3）的各种蛋白质，异种移植物可在体内成像。 </li></ol>

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作者提出的工作有没有利益冲突。

组织重组使用UGSM是一个非常有用的技术来探讨前列腺，导致前列腺癌症的发生的分子事件的发展。已被用于众多应用在前列腺研究中的感应电势UGSM，其中包括提高肿瘤的前列腺细胞系和肿瘤研究上皮间质的相互作用，并形成跨物种前列腺重组体。7,17-20适当的准备UGSM然而，实验成功，将导致污染啮齿动物上皮腺腔形成，可以混淆结果是至关重要的。因此，分离的UGSM从UGSE中（步骤2.4）是?...

为更好的人体模型系统对前列腺癌有一个巨大的需要。尤其是，相关的人体模型系统的正常，非恶性前列腺组织可以操纵基因直接辨别特定基因的作用在前列腺癌的启动将是令人难以置信的信息。基因组时代的来临已经确定了许多基因可能在癌症的形成有一定的作用。然而，缺乏人体实验模型系统严重损害我们的能力，功能测试和表征候选前列腺癌易感基因。一个理想的模型系统，将有利于迅速和更迅速的癌症易感基因组合与适当的转基因啮齿动物模型系统的功能分析。此外，这种人体模型系统将使前列腺癌变的信号机制向发现和验证新疗法的分子特征预防前列腺癌的形成。 人胚胎干细胞（人胚胎干细胞）是能够形成作为异种移植的人类前列腺组织。泰勒等人在2006年报道，人类胚胎干细胞可以诱导在体内与啮齿动物的泌尿生殖窦间充质（UGSM）在8-12周的时间段之内重新组合以形成前列腺上皮细胞。1这些研究是基于以前的工作库尼亚实验室表明，啮齿类动物胚胎UGSM可以促进前列腺分化的干细胞和胚胎在体内的上皮细胞群。2,3前列腺癌的发展从一个胚胎原基称为泌尿生殖窦（UGS），胚胎17天前（E17鼠标一天E18在大鼠）UGS可以删除，并在物理上分为上皮（UGSE）和间充质（UGSM）4本组织重组方法已明显使我们进一步了解前列腺的发育和癌变，特别是光年生长因子和激素信号转导途径和前列腺基质和上皮细胞的分子之间的关系。5-8此方法涉及体外的UGSM组合与干细胞或上皮细胞中的相同或不同的种属，这些细胞/组织的重组体注入和生长经过一段时间的在体内的生长和小鼠异种移植物于主机。4,9，所述植入物包含嵌入在基质组织的前列腺上皮腺状结构。进一步的染色，也可进行，以确定这样的结构是否是真正的前列腺和人类起源。10,11

<table><tbody><tr><td>Hank&amp;rsquo;s Balanced Salt Solution (HBSS)</td><td>GIBCO</td><td>14170</td><td></td></tr><tr><td>DMEM/F12</td><td>GIBCO</td><td>11330</td><td></td></tr><tr><td>R1881</td><td>Sigma</td><td>965-93-5</td><td>Mix to 1 ug/ml in Ethanol (1,000x stock)</td></tr><tr><td>NEAA</td><td>GIBCO</td><td>11140</td><td></td></tr><tr><td>Pen-Strep Solution</td><td>GIBCO</td><td>15070</td><td>100x stock</td></tr><tr><td>Matrigel</td><td>BD Biosciences</td><td>354230</td><td></td></tr><tr><td>KETASET (ketamine hydrochloride) </td><td>Fort Dodge Animal Health</td><td>NDC 0856-2013-01</td><td>100 mg/ml; dilute 1:10 in sterile saline </td></tr><tr><td>AnaSed (xylazine) </td><td>VET-A-MIX, Inc.</td><td>NADA 139-236</td><td>20 mg/ml; dilute 1:10 in sterile saline</td></tr><tr><td>Trypsin</td><td>BD Biosciences</td><td>215240</td><td></td></tr><tr><td>Collagenase </td><td>Sigma</td><td>C2014</td><td></td></tr><tr><td>Ketoprofen </td><td>Fort Dodge</td><td>NDC 0856-4396-01</td><td>100 mg/ml; dilute 1:1,000 in sterile saline</td></tr><tr><td>Altalube eye ointment </td><td>Altaire Pharmaceuticals, Inc.</td><td>NLC 56641-19850</td><td></td></tr><tr><td>Leica MZ16 F Stereomicroscope</td><td>Leica</td><td></td><td>Any good dissecting scope can be used.</td></tr><tr><td>Vannas spring scissors</td><td>Fine Science Tools</td><td>15001-08</td><td></td></tr><tr><td>Syringe</td><td>Hamilton</td><td>84855</td><td></td></tr><tr><td>Hamilton Needle, Small RN, 28 gauge, 0.5inches, Point Style #3 (Blunt)</td><td>Hamilton</td><td>7803-02</td><td>Custom Needle </td></tr><tr><td>Ethanol Prep Pads</td><td>Fisher Scientific</td><td>06-669-62</td><td></td></tr><tr><td>Sterile Gauze Pads</td><td>Fisher Scientific</td><td>22-415-469</td><td></td></tr><tr><td>Ethicon Vicryl Suture (4-0 FS-2)</td><td>MedVet International</td><td>J392H</td><td>Needle-in, dissolvable suture </td></tr><tr><td>Autoclip 9 mm Wound Clips</td><td>Becton Dickenson</td><td>427631</td><td></td></tr><tr><td>PVP Iodine Prep Pads</td><td>Fisher Scientific</td><td>06-669-98</td><td></td></tr><tr><td>Dissector scissor with blunt end</td><td>Fine Science Tools</td><td>14072-10</td><td></td></tr><tr><td>Dumont fine tip forceps</td><td>Fine Science Tools</td><td>11252-50</td><td></td></tr><tr><td>Needle holder with Scissor</td><td>Fine Science Tools</td><td>12002-14</td><td></td></tr></tbody></table>

formation of human prostate epithelium using tissue recombination of rodent urogenital sinus mesenchyme and human stem cells

人源性和激素天真的模型系统，这限制了我们的能力，了解相关前列腺疾病引发的遗传和分子事件的可用性，在前列腺癌的研究进展受到严重限制。对发展更好的模型系统研究人类前列腺癌，我们和其他人利用胚胎鼠的前列腺基质亲前列腺独特的感应电势，被称为泌尿生殖窦间充质（UGSM）。当重组的目标，如胚胎干细胞的多能性的细胞群，UGSM睾酮依赖性诱导正常人类前列腺上皮细胞的形成。这种人体模型系统可用于调查和实验测试候选前列腺癌易感基因的能力比较典型的啮齿动物转基因研究加快。由于人类胚胎干细胞（胚胎）可以在全光照培养转基因克诱导基因表达或siRNA击倒向量组织重组之前，这样的模式有利于测试基因功能的后果，或基因组合，这被认为是促进或防止癌变的。 然而，分离纯人群的UGSM细胞，该技术的挑战和学习往往需要与以前的专业知识的人亲自教导。此外，肾包膜下的细胞混合物接种的免疫功能低下的主机可以是技术上的挑战。这里，我们勾勒，说明适当的隔离的UGSM从啮齿动物胚胎组织混合物形成人类前列腺上皮和肾囊的着床。这样的方法，在目前的阶段，需要在体内异种移植的胚胎干细胞，未来的应用程序可能会包括在体外腺形成或使用诱导式多能性干细胞群（iPS细胞）。

泰勒等人的精彩的 ​​报告基础上，我们的实验室已研制出一种嫁接协议使用常用的H1（NIH-WA01指定，男性基因）人类胚胎干细胞系。1这行已经严格测试，质量控制和核型正常的13，当适当的培养，胚胎干细胞可以保持，扩大，并使用无饲养层培养方法（馈线系统通过干细胞技术市售温哥华BC），冷冻保存在一个未分化和多能状态。14我们协议采用结合与单细胞悬液E...

Watch this Scientific Journal Video about Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells at JoVE.com

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

为了解开最早的分子机制启动前列腺癌，新颖创新的人体模型系统和方法是迫切需要的。诱导多能干细胞群形成的人类前列腺上皮细胞的潜力预前列腺泌尿生殖窦间充质（UGSM）是一个强大的实验工具，在前列腺研究。

人类前列腺上皮细胞形成的使用鼠类泌尿生殖窦间质细胞和人类干细胞的组织重组

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

formation-human-prostate-epithelium-using-tissue-recombination-rodent

Research

JoVE Journal

Medicine

13.6K 次观看.  University of Chicago. 为了解开最早的分子机制启动前列腺癌，新颖创新的人体模型系统和方法是迫切需要的。诱导多能干细胞群形成的人类前列腺上皮细胞的潜力预前列腺泌尿生殖窦间充质（UGSM）是一个强大的实验工具，在前列腺研究。

视频: Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Identification, Histological Characterization, and Dissection of Mouse Prostate Lobes for In Vitro 3D Spheroid Culture Models

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate cancer (PCa). Understanding the anatomy and histology of the mouse prostate is important for the efficient use and proper characterization of such animal models. The mouse prostate has four distinct pairs of lobes, each with their own characteristics. This article demonstrates the proper method of dissection and identification of mouse prostate lobes for disease analysis. Post-dissection, the prostate cells can be further cultured in vitro for mechanistic understanding. Since mouse prostate primary cells tend to lose their normal characteristics when cultured in vitro, we outline here a method for isolating the cells and growing them as 3D spheroid cultures, which is effective for preserving the physiological characteristics of the cells. These 3D cultures can be used for analyzing cell morphology and behavior in near-physiological conditions, investigating altered levels and localizations of key proteins and pathways involved in the development and progression of a disease, and looking at responses to drug treatments.

Genetically engineered mice are useful models for investigating prostate cancer mechanisms. Here we present a protocol to identify and dissect prostate lobes from a mouse urogenital system, differentiate them based on histology, and isolate and culture the primary prostate cells in vitro as spheroids for downstream analyses.

体外3D 球形培养模型小鼠前列腺叶的鉴定、组织学特征及解剖

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate ...

科研

癌症研究

Evaluating the Differentiation Capacity of Mouse Prostate Epithelial Cells Using Organoid Culture

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation capacity of mouse prostate basal and luminal cells during development, tissue-regeneration and transformation. However, evaluating cell-intrinsic and extrinsic regulators of prostate epithelial differentiation capacity using a lineage tracing approach often requires extensive breeding and can be cost-prohibitive. In the prostate organoid assay, basal and luminal cells generate prostatic epithelium ex vivo. Importantly, primary epithelial cells can be isolated from mice of any genetic background or mice treated with any number of small molecules prior to, or after, plating into three-dimensional (3D) culture. Sufficient material for evaluation of differentiation capacity is generated after 7-10 days. Collection of basal-derived and luminal-derived organoids for (1) protein analysis by Western blot and (2) immunohistochemical analysis of intact organoids by whole-mount confocal microscopy enables researchers to evaluate the ex vivo differentiation capacity of prostate epithelial cells. When used in combination, these two approaches provide complementary information about the differentiation capacity of prostate basal and luminal cells in response to genetic or pharmacological manipulation.

Mouse prostate organoids represent a promising context to evaluate mechanisms that regulate differentiation. This paper describes an improved approach to establish prostate organoids, and introduces methods to (1) collect protein lysate from organoids, and (2) fix and stain organoids for whole-mount confocal microscopy.

利用有机体培养评价小鼠前列腺皮细胞的分化能力

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation ...

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident stem cells are relatively quiescent with niche restraints in adult tissues, they enter the cell cycle in anchor-free three-dimensional (3D) culture and undergo both symmetric and asymmetric cell division, giving rise to both stem and progenitor cells. The latter proliferate rapidly and are the major cell population at various stages of lineage commitment, forming heterogeneous spheroids. Using primary normal human prostate epithelial cells (HPrEC), a spheroid-based, label-retention assay was developed that permits the identification and functional isolation of the spheroid-initiating stem cells at a single cell resolution.
HPrEC or cell lines are two-dimensionally (2D) cultured with BrdU for 10 days to permit its incorporation into the DNA of all dividing cells, including self-renewing stem cells. Wash out commences upon transfer to the 3D culture for 5 days, during which stem cells self-renew through asymmetric division and initiate spheroid formation. While relatively quiescent daughter stem cells retain BrdU-labeled parental DNA, the daughter progenitors rapidly proliferate, losing the BrdU label. BrdU can be substituted with CFSE or Far Red pro-dyes, which permit live stem cell isolation by FACS. Stem cell characteristics are confirmed by in vitro spheroid formation, in vivo tissue regeneration assays, and by documenting their symmetric/asymmetric cell divisions. The isolated label-retaining stem cells can be rigorously interrogated by downstream molecular and biologic studies, including RNA-seq, ChIP-seq, single cell capture, metabolic activity, proteome profiling, immunocytochemistry, organoid formation, and in vivo tissue regeneration. Importantly, this marker-free functional stem cell isolation approach identifies stem-like cells from fresh cancer specimens and cancer cell lines from multiple organs, suggesting wide applicability. It can be used to identify cancer stem-like cell biomarkers, screen pharmaceuticals targeting cancer stem-like cells, and discover novel therapeutic targets in cancers.

Using human primary prostate epithelial cells, we report a novel biomarker-free method of functional characterization of stem-like cells by a spheroid-based label-retention assay. A step-by-step protocol is described for BrdU, CFSE, or Far Red 2D cell labeling; three-dimensional spheroid formation; label-retaining stem-like cell identification by immunocytochemistry; and isolation by FACS.

从3维球体培养中分离出类似干细胞的细胞

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident ...

Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign Prostatic Hyperplasia

New therapies for two common prostate diseases, prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend critically on experiments evaluating their hormonal regulation. Sex steroid hormones (notably androgens and estrogens) are important in PrCa and BPH; we probe their respective roles in inducing prostate growth and carcinogenesis in mice with experiments using compressed hormone pellets. Hormone and/or drug pellets are easily manufactured with a pellet press, and surgically implanted into the subcutaneous tissue of the male mouse host. We also describe a protocol for the evaluation of hormonal carcinogenesis by combining subcutaneous hormone pellet implantation with xenografting of prostate cell recombinants under the renal capsule of immunocompromised mice. Moreover, subcutaneous hormone pellet implantation, in combination with renal capsule xenografting of BPH tissue, is useful to better understand hormonal regulation of benign prostate growth, and to test new therapies targeting sex steroid hormone pathways.

We describe the manufacture of compressed hormone pellets, as well as subcutaneous surgical implantation into mice. This strategy can be combined with the growth of cell and tissue xenografts under the renal capsule of athymic nude mice to evaluate hormonal carcinogenesis and regulation of benign prostate growth.

肾小囊和异种移植皮下颗粒植入前列腺癌发生和良性前列腺增生症的评价

New therapies for two common prostate diseases,  prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend  critically on experiments ...

医学

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

上皮细胞复育和肾组织的发展准备啮齿动物细胞外基质的支架

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

生物工程

Handling and Assessment of Human Primary Prostate Organoid Culture

This paper describes a detailed protocol for three-dimensional (3D) culturing, handling, and evaluation of human primary prostate organoids. The process involves seeding of epithelial cells sparsely in a 3D matrix gel on a 96-well microplate with media changes to cultivate expansion into organoids. Morphology is then assessed by whole-well capturing of z-stack images. Compression of z-stacks creates a single in-focus image from which organoids are measured to quantify a variety of outputs, including circularity, roundness, and area.DNA, RNA, and protein can be collected from organoids recovered from the matrix gel. Cell populations of interest can be assessed by organoid dissociation and flow cytometry. Formalin-fixation-paraffin-embedding (FFPE) followed by sectioning is used for the histological assessment and antibody staining. Whole-mount immunofluorescent staining preserves organoid morphology and facilitates observation of protein localization in organoids in situ. Commercial assays that are traditionally used for 2D monolayer cells can be modified for 3D organoids. Used together, the techniques in this protocol provide a robust toolbox to quantify prostate organoid growth, morphologic characteristics, and expression of differentiation markers.

Here, we present a protocol to guide human primary prostate organoid handling then suggest endpoints to assess phenotype. Seeding, culture maintenance, recovery from matrix gel, morphologic quantification, embedding and sectioning, FFPE sectioning, whole-mount staining, and application of commercial assays are described.

人原发性前列腺组织培养的处理与评价

This paper describes a detailed protocol for three-dimensional (3D) culturing, handling, and evaluation of human primary prostate organoids. The process ...

Murine Prostate Micro-dissection and Surgical Castration

Mouse models are used extensively to study prostate cancer and other diseases. The mouse is an excellent model with which to study the prostate and has been used as a surrogate for discoveries in human prostate development and disease. Prostate micro-dissection allows consistent study of lobe-specific prostate anatomy, histology, and cellular characteristics in the absence of contamination of other tissues. Testosterone affects prostate development and disease. Androgen deprivation therapy is a common treatment for prostate cancer patients, but many prostate tumors become castration-resistant. Surgical castration of mouse models allows for the study of castration resistance and other facets of hormonal biology on the prostate. This procedure can be coupled with testosterone reintroduction, or hormonal regeneration of the prostate, a powerful method to study stem cell lineages in the prostate. Together, prostate micro-dissection and surgical castration opens up a multitude of opportunities for robust and consistent research of prostate development and disease. This manuscript describes the protocols for prostate micro-dissection and surgical castration in the laboratory mouse.

This manuscript describes the protocols for prostate micro-dissection and surgical castration in the laboratory mouse. We also depict representative results produced by these protocols. Finally, we discuss the advantages and utilization of these protocols.

鼠前列腺微解剖和手术去势

Mouse models are used extensively to study prostate cancer and other diseases. The mouse is an excellent model with which to study the prostate and has ...

使用胶原支架进行猪单阶段尿流改道手术

Surgical Model for Single-Staged Tissue-Engineered Urothelial Tubes in Minipigs

Reconstructive surgeries are often challenged by a lack of grafting tissue. In the treatment of urogenital malformations, the conventional solution has been harvesting gastrointestinal tissue for non-orthotopic reconstruction due to its abundance to reestablish normal function in the patient. The clinical outcomes after rearranging native tissues within the body are often associated with significant morbidity; thus, tissue engineering holds specific potential within this field of surgery. Despite substantial advances, tissue-engineered scaffolds have not yet been established as a valid surgical treatment alternative, mainly due to the costly and complex requirements of materials, production, and implantation. In this protocol, we present a simple and accessible collagen-based tubular scaffold embedded with autologous organ-specific tissue particles, designed as a conduit for urinary diversion. The scaffold is constructed during the primary surgical procedure, comprises commonly available surgical materials, and requires conventional surgical skills. Secondly, the protocol describes an animal model designed to evaluate the short-term in vivo outcomes post-implantation, with the possibility of additional variations to the procedure. This publication aims to demonstrate the procedure step-by-step, with special attention to the use of autologous tissue and a tubular form.

作者聚焦:改善先天性畸形和后天性疾病重建手术结果的创新方法

Tissue-engineered implants for reconstructive surgery rarely progress beyond preclinical trials due to laborious ex vivo culturing, which includes complex and expensive scaffold components. Here, we present a single-staged procedure designed for urinary diversion with an accessible collagen-based tubular scaffold containing autologous micrografts.

小型猪单期组织工程尿路上皮管手术模型

Reconstructive surgeries are often challenged by a lack of grafting tissue. In the treatment of urogenital malformations, the conventional solution has ...

人类前列腺上皮细胞形成的使用鼠类泌尿生殖窦间质细胞和人类干细胞的组织重组

摘要

探索更多视频

此视频中的章节

体外3D 球形培养模型小鼠前列腺叶的鉴定、组织学特征及解剖

利用有机体培养评价小鼠前列腺皮细胞的分化能力

从3维球体培养中分离出类似干细胞的细胞

肾小囊和异种移植皮下颗粒植入前列腺癌发生和良性前列腺增生症的评价

上皮细胞复育和肾组织的发展准备啮齿动物细胞外基质的支架

人原发性前列腺组织培养的处理与评价

鼠前列腺微解剖和手术去势

小型猪单期组织工程尿路上皮管手术模型

小型猪单期组织工程尿路上皮管手术模型

小型猪单期组织工程尿路上皮管手术模型