Name: a rapid filter insert-based 3d culture system for primary prostate cell differentiation
Uploaded: 2017-02-13T15:00:00.000+00:00
Duration: 9 min 23 s
Description: Watch this Scientific Journal Video about A Rapid Filter Insert-based 3D Culture System for Primary Prostate Cell Differentiation at JoVE.com

这项研究是由T32（CA 9686-18）和TL1（TL1TR001431）博士后培训给予奖励（LT），DOD PC140268（CA），W81XWH-13-1-0327（CA）TR000102-04（CA），以及支持U01 PAR-12-095（库马尔）和P30 CA051008-21（韦纳）。样品固定，切片和染色是在隆巴迪综合癌症中心的组织学与组织共享资源进行。我们感谢理查德·施莱格尔有益的讨论。内容完全是作者的责任，并不一定代表美国国家癌症研究所和美国国立卫生研究院的官方意见。

1.三维细胞培养建立插入系统<ol><li>放置在倒置方向（滤波器侧上）的6孔板（ 图1A）向下2聚碳酸酯细胞培养插入。 </li><li>在水施加0.1％明胶的薄层于刀片的底侧并晾干（10-20分钟）在生物安全柜以保持无菌。 </li><li>重复步骤1.2，共上刀片0.1％明胶的3应用程序两次以上。 注:明胶涂层将有助于室之间的限制扩散。 </li><li>为了收集从标准培养条件的主要社区康复中心，加入5毫升的PBS洗培养瓶中。 <ol><li> Trypsinize使用细胞（1毫升为一个T-25和2mL的T-75）0.25％胰蛋白酶-EDTA在37℃5分钟。轻轻拍打烧瓶侧面中撞出的细胞提供帮助。然后继续以停止胰蛋白酶反应，并通过添加完全DMEM的5毫升收集细胞。 </li><li>搜集<html>将细胞在15毫升的管中。离心管中，在4℃和188 XG和使用自动细胞计数器或血细胞计数器计数。 </li><li>重悬在250微升的条件培养基（CM）60万的CRC，并添加到正确定向（滤波器面朝下）培养插入物（ 图1B）。这被称为内室。 注意:CM是F-媒体通过照射J2细胞的条件和包含10微米Y-27632如前所述7，8，9，11。 </li></ol></li><li>应用2毫升的CM到6孔板的外室，并在37℃下孵育过夜（至少18小时）。 </li><li>小心地用一个1毫升或200微升吸管除去在CM上的内室和缓慢分化培养基（DM）中添加用1毫升吸移管内室满为止。小心不要扰乱细胞层。 </li></ol>乐"&gt; 2，3D文化的维护<ol><li>检查文化的每一天。健康的细胞出现， 如图2。 </li><li>改变每天或每隔一天取决于细胞的代谢中的外室中的CM。 </li><li>当内腔室改变颜色（黄色）的媒体，小心地取出在内部过滤器室用1毫升或200微升吸管介质。 </li><li>吸媒体在外6孔板室与吸气提示。 </li><li>用1毫升吸管，新鲜DM慢慢地应用到内室满为止。要小心，不要刮或以其他方式打跑细胞层。 </li><li>用2mL新鲜的CM代替在外部腔室中的介质。 </li><li>维持培养2周，然后进行处理所需的双分子隔离。 注意:在6孔板中的每一行，共六个刀片（ 图1B），应每实验处理，以获得足够的细胞DNA，RNA或蛋白质进行分析。 </li><li>在外部腔室抽吸介质，并用2毫升磷酸盐缓冲盐水（PBS）冲洗。 </li><li>小心地从用1毫升或200微升吸管内腔取出介质，并添加500微升PBS中。避免刮擦或以其它方式干扰在膜上的细胞。 </li><li>从外室抽吸PBS中并小心地从内室除去的PBS与1毫升或200微升吸管。一旦PBS已被去除，直接进行以下详述的适当的应用程序。不要让膜干燥。文化可以保持简单的PBS，直到应用程序已经准备好开始。 </li></ol> 3.加工球团银行业的过滤器<ol><li> 100μL的0.25％胰蛋白酶-EDTA添加到每个内室，并在37℃下孵育5分钟。 </li><li>简单地说，仔细搅拌/刮膜表面的细胞用200微升吸管吸取，同时向上和向下（避免PUNC图灵膜）。在37℃下孵育1分钟。 </li><li>添加的CM 250微升到第一内室和吸取上下轻轻冲洗过滤器。 </li><li>转移悬浮细胞至包含相同的媒体的条件并重复上下吹打相邻过滤室。 </li><li>重复此过程为每个单一条件的刀片。收集并转移细胞到1.5mL离心管中。 </li><li>每次冲洗内腔与CM额外100-200微升收集任何剩余的细胞。添加到1.5毫升离心管中，在步骤3.5。 </li><li>旋在423 xg离心所述细胞在微量在4℃下5分钟。 </li><li>吸出上清液并用1000微升的PBS洗一次细胞沉淀。 </li><li>在423 xg离心再次旋在微量在4℃下5分钟。 </li><li>抽吸PBS中并在-80℃以备后用冷冻。 </li></ol> 4.处理RNA或DNA分离过滤器<ol&gt; <li>添加硫氰酸胍 - 苯酚 - 氯仿或类似的提取试剂的200μL到内室和通过上下吹打简要地搅动膜表面上的细胞。 </li><li> 孵育在提取试剂，在室温下5分钟与外室干。 <ol><li>作为过滤器可以从插入件分离，通过上下吹打提取液洗解除关联过滤膜。通过倾斜6孔板并且吸取残留液体收集尽可能多的提取试剂成为可能。 </li></ol></li><li>按照制造商的净化和DNA，RNA或蛋白回收协议。 </li></ol> 5.处理蛋白质分离过滤器<ol><li>放置在冰上6孔板的过滤器插入件。到内室中，添加10微升补充有1mM的氟化钠（氟化钠），1mM的钒酸钠（NAV），100毫dithiothre蛋白质裂解缓冲液itol醇（DTT）和100微升抗蛋白酶鸡尾酒。 </li><li>搅动/刮去膜表面上的细胞用20微升吸管而上下吹吸和。避免在裂解液产生气泡。 </li><li>孵育滤芯在冰上6孔板10分钟。 </li><li>重复刮，然后用清水冲洗裂解液膜表面。 </li><li>从6插入收集裂解物并转移到1.5mL离心管在冰上。 </li><li>与另外的10μL的裂解缓冲液并转移到下一个过滤器冲洗所述第一膜。 </li><li>重复步骤5.6所有插入，收集任何残留的裂解缓冲液，并加入到1.5mL管中（步骤5.5）。 </li><li>在冰上孵育样品额外5分钟。 </li><li>自旋以最大速度（16873 XG在台式离心机）在4℃下15分钟。 </li><li>吸取裂解上清液到一个新的1.5毫升管。 </li><li>继续蛋白浓度或申通快递的分析再在-80℃的裂解液。 </li></ol> 6.处理H＆E和免疫染色<ol><li> 500微升和1毫升10％NBF（中性缓冲的福尔马林）添加到内和外室，并让在4℃孵育过夜。 </li><li>从外室吸出NBF并添加1毫升羟乙基琼脂糖（HEA）处理凝胶到1.5mL离心。使用低功率慢慢融化琼脂糖在微波炉和反复10-20小号脉冲直到琼脂糖融化。 </li><li>保持HEA在37℃的温水澡，防止凝固，直到准备使用。 </li><li>从内腔中取出的NBF和应用熔融HEA的25μL到内室，并允许琼脂糖固化2-5分钟。 </li><li>在10％NBF湿2泡沫垫组织学和放一个垫在包埋盒（ 见图3D）。 </li><li>使用＃11刀片的手术刀（其具有非常尖锐的，细尖头刀）从刀片的底侧得分滤波器腔室，部分地从塑料插入腔室释放它。 </li><li>放置NBF少量（100-200微升）在培养皿中并浸泡在NBF（ 图3A）的部分移出过滤器。 </li><li>使用＃10刀片的手术刀，轻轻压靠在过滤器的中间，从插入室的内部，以充分去除从插入筒（ 图3B）的滤波器。如果存在仍连接到筒的过滤器的任何部分，用手术刀断绝它。 </li><li>切HEA涂层过滤器的一半与＃10手术刀刀片（ 图3C）。 </li><li>过滤器的每一半放置到在步骤6.4（ 图3D）中制备的组织学暗盒中泡沫垫。 </li><li>第二个10％NBF浸泡海绵添加到盒，夹在中间的滤光器。 </li><li>捕捉封住NBF纸盒，地点和孵育过夜。 </li><li>流程过滤器石蜡块切片和染色以前12描述。 </li></ol>

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原代细胞系是癌症研究的一个重要的，迅速发展的平台。基于插入培养三维培养系统支持原发性前列腺CRC的为期两周的时间内的分化。该CRC滤波方法代表前列腺研究一个新的，中等吞吐量方法。现有鼠标PDX模型是耗时且极其昂贵，并且许多对PDX样品不能在培养物中生长，限制研究者发起实验和它们的用途。此外，同时建立PDX的成功率相差很大13中，CRC方法具有成功的建立细胞系<s...

这是个性化的个人识别和使用的癌症疗法在癌症研究的一个主要目标。最近，新的方法已经开发了允许在建立原代细胞培养物的更易于，潜在地同时提供识别和检验的个性化疗法的方法。例如，在基于R-脊椎前列腺类器官的方法1可用于三维（3D）正常和转移性前列腺癌的细胞在商业细胞外基质的培养（ 例如 ，基质胶），而细胞的有条件重新编程（CRC）的方法在乔治城2开发，利用3以上标准的2D培养条件。具体而言，Rho激酶抑制剂（Y-27632）和辐照J2小鼠成纤维细胞饲养细胞的组合导致角质形成细胞的CRC 2的无限期培养。在CRC的方法是极为坚固，与原代细胞系成功建立，并从前列腺和其他许多正常和恶性上皮组织3无限期地保持下去。重要的是，允许在谁没有一些以前的药物治疗的患者复发性呼吸道乳头状病原学基础的快速识别我们的CRC技术。此外，使用正常和肿瘤来源的CRC，FDA的成功鉴定批准的药物，伏立诺他，二周初始组织活检的内作出。病人被放置在伏立诺他，导致他们的疾病4的成功治疗。 正常的前列腺是由鲁米那，基底和罕见的神经内分泌细胞5。管腔细胞形成腺体的柱状上皮层和表达雄激素受体（AR），以及其他管腔标记物如角蛋白8和18和亲状态特定抗原（PSA）6。相反，基底细胞是局部管腔层下方，表达细胞角蛋白5和p63，但AR 5的低水平。我们7，8，9和其他10已成功地应用于前列腺康复中心在临床前机械药敏调查。然而，当标准的2D组织培养条件下生长，这些细胞不能完全啮合AR信令10。重要的是，当免疫缺陷小鼠的肾包膜下放置两个CRC恢复正常前列腺腺体结构和功能说明放置在一个宽容的环境，当前列腺康复中心保留其血统的承诺。此处所描述的基于插入滤波器的细胞培养系统的发展允许快速（2周） 在体外的前列腺CRC的分化所证明by中的Ar和Ar靶基因的表达增加以及降低p63的水平。 使用的过滤器插入件包含聚碳酸酯膜（孔径0.4微米），可支持的哺乳动物细胞的培养。该系统，为开发，利用正常的和恶性的前列腺康复中心，6孔培养皿和滤芯的。通过J2单元11调节的细胞培养基被放置在底室和前列腺区分媒体在顶室中。本文描述的技术支持2周的时间内前列腺管腔细胞分化，与个性化药物的目标相一致。它也必须开发出允许培养物的全面分子，遗传和细胞分析的方法。用于从过滤表面释放的细胞中分离DNA，RNA和蛋白质的方法已被开发并简化为准确重复样品的处理。菲娜LLY，需要取下过滤网，使嵌入，切片和H＆E，免疫组化及免疫荧光染色的方法，充分说明。

<table><tbody><tr><td>Corning Costar Snapwell Culture Inserts</td><td>Corning</td><td>3801</td><td></td></tr><tr><td>Millicell Cell Culture Inserts</td><td>Millipore</td><td>PIHP01250</td><td></td></tr><tr><td>Multiwell 6 Well</td><td>Falcon</td><td>353046</td><td></td></tr><tr><td>Gelatin 0.1% in water</td><td>Stemcell Technologies</td><td>7903</td><td></td></tr><tr><td>Sterile Saftey Scapel 10 Blade</td><td>Integra Miltex</td><td>4-510</td><td></td></tr><tr><td>Sterile Standard Scalpel 11 Blade</td><td>Integra Miltex</td><td>4411</td><td></td></tr><tr><td>RIPA Lysis and Extraction Buffer</td><td>Thermofisher Scientific</td><td>89900</td><td></td></tr><tr><td>Sodium Fluoride</td><td>Fishcer Scientific</td><td>S299-100</td><td></td></tr><tr><td>Sodium Vanadate</td><td>Fishcer Scientific</td><td>13721-39-6</td><td></td></tr><tr><td>Dithiothreitol</td><td>Sigma-Aldrich</td><td>3483123</td><td></td></tr><tr><td>Protease Inhibitor Cocktail (AEBSF, Aprotinin, Bestatin, E-64, Leupeptin and Pepstatin A)</td><td>Sigma-Aldrich</td><td>P8340</td><td></td></tr><tr><td>0.25% Trypsin-EDTA (1x)</td><td>Thermofisher Scientific</td><td>25200-056</td><td></td></tr><tr><td>DMEM</td><td>Thermofisher Scientific</td><td>11965-092</td><td></td></tr><tr><td>FBS</td><td>Sigma-Aldrich</td><td>F2442</td><td></td></tr><tr><td>Glutamine</td><td>Thermofisher Scientific</td><td>25030081&amp;nbsp;</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>Thermofisher Scientific</td><td>15140-122</td><td></td></tr><tr><td>F-12 Nutrient Mix Media</td><td>Thermofisher Scientific</td><td>11765054</td><td></td></tr><tr><td>Y-27632 dihydrochloride Rock inhibitor</td><td>Enzo</td><td>ALX-270-333-M025</td><td></td></tr><tr><td>Hydrocortisone</td><td>Sigma-Aldrich</td><td>H0888</td><td></td></tr><tr><td>EGF</td><td>Thermofisher Scientific</td><td>PHG0315</td><td></td></tr><tr><td>Insulin</td><td>Thermofisher Scientific</td><td>12585-014</td><td></td></tr><tr><td>Cholera Toxin</td><td>Sigma-Aldrich</td><td>C3012</td><td></td></tr><tr><td>Gentamicin</td><td>Thermofisher Scientific</td><td>15710-064</td><td></td></tr><tr><td>Fungizone</td><td>Fisher Scientific</td><td>BP264550</td><td></td></tr><tr><td>Trizol Reagent</td><td>Invitrogen</td><td>15596026</td><td></td></tr><tr><td>Histogel Specimen Processing Gel (hydroxyethyl agarose (HEA))</td><td>Thermo Scientific</td><td>HG-4000-012</td><td></td></tr><tr><td>Non-Adherent Dressing</td><td>Telfa</td><td>KDL2132Z</td><td></td></tr><tr><td>Cell Culture Dish</td><td>Sigma</td><td>SIAL0167</td><td></td></tr><tr><td>HISTOSETTE II Tissue Processing / Embedding Cassettes</td><td>Crystalgen</td><td>CG-M492</td><td></td></tr></tbody></table>

a rapid filter insert-based 3d culture system for primary prostate cell differentiation

有条件地重新编程细胞（CRC）的为原代细胞培养，并制定患者的细胞系广泛的"活生物银行"的能力，可持续发展的方法。对于许多类型的上皮细胞，各种三维（3D）培养的方法已经被描述了支持改进的分化状态。虽然结直肠癌保留他们的后裔承诺从他们被隔离的组织，他们无法在正常二维（2D）培养条件下生长，以表达许多与起源组织相关的分化标志。以增强患者来源CRC的前列腺癌研究中的应用，三维培养格式已被定义，它使一个快速（2周总）管腔细胞在正常和肿瘤衍生的前列腺上皮细胞的分化。这里，基于插入滤波器格式为正常和恶性前列腺CRC的培养和分化说明。一德提供的细胞收集和处理的免疫组化及免疫荧光染色所需的程序尾描述。总体描述的3D格式的文化，与主CRC线相结合，提供了重要的中期和高通量的模型系统基于生物样本前列腺的研究。

细胞培养插管的系统是用于产生支持管腔细胞分化前列腺CRC的3D培养相对简单和快速的过程。该系统的原理图（ 图1A）突出明胶涂层的过滤器插入件的底表面上的应用。该刀片只为推翻明胶的应用。在图1B中的过滤器是在用于培养适当的取向。使用透明细胞插入物，一个健康的三维前列腺CRC文化的代表图像显示（10X）（ 图2）。健康CRC?...

Watch this Scientific Journal Video about A Rapid Filter Insert-based 3D Culture System for Primary Prostate Cell Differentiation at JoVE.com

A Rapid Filter Insert-based 3D Culture System for Primary Prostate Cell Differentiation

Here, we present a method for the establishment of a rapid in vitro system that supports the three dimensional culturing and subsequent luminal differentiation of primary prostate epithelial cells.

基于插入 - 滤池3D文化系统原发性前列腺细胞分化

Conditionally reprogrammed cells (CRCs) provide a sustainable method for primary cell culture and the ability to develop extensive &#34;living ...

a-rapid-filter-insert-based-3d-culture-system-for-primary-prostate

Universidad San Sebastian

Research

JoVE Journal

Cancer Research

8.5K 次观看.  Lombardi Comprehensive Cancer Center. Here, we present a method for the establishment of a rapid in vitro system that supports the three dimensional culturing and subsequent luminal differentiation of primary prostate epithelial cells.

视频: A Rapid Filter Insert-based 3D Culture System for Primary Prostate Cell Differentiation

How to Study Basement Membrane Stiffness as a Biophysical Trigger in Prostate Cancer and Other Age-related Pathologies or Metabolic Diseases

Here we describe a protocol that can be used to study the biophysical microenvironment related to increased thickness and stiffness of the basement membrane (BM) during age-related pathologies and metabolic disorders (e.g. cancer, diabetes, microvascular disease, retinopathy, nephropathy and neuropathy). The premise of the model is non-enzymatic crosslinking of reconstituted BM (rBM) matrix by treatment with glycolaldehyde (GLA) to promote advanced glycation endproduct (AGE) generation via the Maillard reaction. Examples of laboratory techniques that can be used to confirm AGE generation, non-enzymatic crosslinking and increased stiffness in GLA treated rBM are outlined. These include preparation of native rBM (treated with phosphate-buffered saline, PBS) and stiff rBM (treated with GLA) for determination of: its AGE content by photometric analysis and immunofluorescent microscopy, its non-enzymatic crosslinking by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) as well as confocal microscopy, and its increased stiffness using rheometry. The procedure described here can be used to increase the rigidity (elastic moduli, E) of rBM up to 3.2-fold, consistent with measurements made in healthy versus diseased human prostate tissue. To recreate the biophysical microenvironment associated with the aging and diseased prostate gland three prostate cell types were introduced on to native rBM and stiff rBM: RWPE-1, prostate epithelial cells (PECs) derived from a normal prostate gland; BPH-1, PECs derived from a prostate gland affected by benign prostatic hyperplasia (BPH); and PC3, metastatic cells derived from a secondary bone tumor originating from prostate cancer. Multiple parameters can be measured, including the size, shape and invasive characteristics of the 3D glandular acini formed by RWPE-1 and BPH-1 on native versus stiff rBM, and average cell length, migratory velocity and persistence of cell movement of 3D spheroids formed by PC3 cells under the same conditions. Cell signaling pathways and the subcellular localization of proteins can also be assessed.

Here we explain a protocol for modelling the biophysical microenvironment where crosslinking and increased stiffness of the basement membrane (BM) induced by advanced glycation endproducts (AGEs) has pathological relevance.

如何学习基底膜刚度生物物理触发在前列腺癌和其他与年龄相关的病理或代谢性疾病

Here we describe a protocol that can be used to study the biophysical microenvironment related to increased thickness and stiffness of the basement ...

科研

癌症研究

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 ...

Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) are a rigorous method for studying mammalian development and disease. Our laboratory has developed several GEMMs of prostate cancer (PCa) that lack expression of one or multiple tumor suppressor genes using the site-specific Cre-loxP recombinase system and a prostate-specific promoter. In this article, we describe our method for necropsy of these PCa GEMMs, primarily focusing on dissection of mouse prostate tumors. New methods developed over the last decade have facilitated the culture of epithelial-derived cells to model organ systems in vitro in three dimensions. We also detail a 3D cell culture method to generate tumor organoids from mouse PCa GEMMs. Pre-clinical cancer research has been dominated by 2D cell culture and cell line-derived or patient-derived xenograft models. These methods lack tumor microenvironment, a limitation of using these techniques in pre-clinical studies. GEMMs are more physiologically-relevant for understanding tumorigenesis and cancer progression. Tumor organoid culture is an in vitro model system that recapitulates tumor architecture and cell lineage characteristics. In addition, 3D cell culture methods allow for growth of normal cells for comparison to tumor cell cultures, rarely possible using 2D cell culture techniques. In combination, use of GEMMs and 3D cell culture in pre-clinical studies has the potential to improve our understanding of cancer biology.&#160;

We show a method for necropsy and dissection of mouse prostate cancer models, focusing on prostate tumor dissection. A step-by-step protocol for generation of mouse prostate tumor organoids is also presented.

从前列腺癌的遗传工程小鼠模型生成肿瘤有机体

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) ...

遗传学

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 ...

Prostate Organoid Cultures as Tools to Translate Genotypes and Mutational Profiles to Pharmacological Responses

Presented here is a protocol to study pharmacodynamics, stem cell potential, and cancer differentiation in prostate epithelial organoids. Prostate organoids are androgen responsive, three-dimensional (3D) cultures grown in a defined medium that resembles the prostatic epithelium. Prostate organoids can be established from wild-type and genetically engineered mouse models, benign human tissue, and advanced prostate cancer. Importantly, patient derived organoids closely resemble tumors in genetics and in vivo tumor biology. Moreover, organoids can be genetically manipulated using CRISPR/Cas9 and shRNA systems. These controlled genetics make the organoid culture attractive as a platform for rapidly testing the effects of genotypes and mutational profiles on pharmacological responses. However, experimental protocols must be specifically adapted to the 3D nature of organoid cultures to obtain reproducible results. Described here are detailed protocols for performing seeding assays to determine organoid formation capacity. Subsequently, this report shows how to perform drug treatments and analyze pharmacological response via viability measurements, protein isolation, and RNA isolation. Finally, the protocol describes how to prepare organoids for xenografting and subsequent in vivo growth assays using subcutaneous grafting. These protocols yield highly reproducible data and are widely applicable to 3D culture systems.

Presented here is a protocol to study pharmacological responses in prostate epithelial organoids. Organoids closely resemble in vivo biology and recapitulate patient genetics, making them attractive model systems. Prostate organoids can be established from wildtype prostates, genetically engineered mouse models, benign human tissue, and advanced prostate cancer.

前列腺有机体培养作为工具，将基因型和突变谱转化为药理反应

Presented here is a protocol to study pharmacodynamics, stem cell potential, and cancer differentiation in prostate epithelial organoids. Prostate ...

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 ...

Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred to as patient-derived organoids (PDO), are an invaluable resource for studying the mechanism of tumorigenesis and metastasis of prostate cancer. Their main advantage is that they maintain the distinctive genomic and functional heterogeneity of the original tissue compared to conventional cell lines that do not. Furthermore, 3D cultures of PDO can be used to predict the effects of drug treatment on individual patients and are a step towards personalized medicine. Despite these advantages, few groups routinely use this method in part because of the extensive optimization of PDO culture conditions that may be required for different patient samples. We previously demonstrated that our prostate cancer bone metastasis PDX model, PCSD1, recapitulated the resistance of the donor patient&rsquo;s bone metastasis to anti-androgen therapy. We used PCSD1 3D organoids to characterize further the mechanisms of anti-androgen resistance. Following an overview of currently published studies of PDX and PDO models, we describe a step-by-step protocol for 3D culture of PDO using domed or floating basement membrane (e.g., Matrigel) spheres in optimized culture conditions. In vivo stitch imaging and cell processing for histology are also described. This protocol can be further optimized for other applications including western blot, co-culture, etc. and can be used to explore characteristics of 3D cultured PDO pertaining to drug resistance, tumorigenesis, metastasis and therapeutics.

Establishment and Analysis of Three-Dimensional 3D Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional cultures of patient BMPC specimens and xenografts of bone metastatic prostate cancer maintain the functional heterogeneity of their original tumors resulting in cysts, spheroids and complex, tumor-like organoids. This manuscript provides an optimization strategy and protocol for 3D culture of heterogeneous patient derived samples and their analysis using IFC.

从患者前列腺癌骨转移标本及其异种移植物中提取的三维（3D）有机物的建立与分析

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred ...

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 ...

生物工程

A Triple Culture Cell System Modeling the Human Blood-Brain Barrier

The delivery of drugs to the brain remains a challenge due to the blood-brain barrier's (BBB) highly specific and restrictive properties, which controls and restrict access to the brain parenchyma. However, with the development of nanotechnologies, large panels of new nanomaterials were developed to improve drug delivery, highlighting the need for reliable in vitro microsystems to predict brain penetration in the frame of preclinical assays. Here is a straightforward method to set up a microphysiological system to model the BBB using solely human cells. In its configuration, the model consists of a triple culture including brain-like endothelial cells (BLECs), pericytes, and astrocytes, the three main BBB cellular actors necessary to induce and regulate the BBB properties in a more physiological manner without the requirement of tightening compounds. The model developed in a 12-well plate format, ready after 6 days of triple culture, is characterized in physical properties, gene, and protein expressions and used for polymeric nanogel transport measurement. The model can be used for an extensive range of experiments in healthy and pathological conditions and represents a valuable tool for preclinical assessments of molecule and particle transport, as well as inter-and intracellular trafficking.

This protocol describes a method to establish a human blood-brain barrier (BBB) in vitro model. The endothelial cells and pericytes are seeded on each side of an insert filter (blood compartment), and astrocytes are seeded in the bottom well (brain compartment). The model characterized was used for nanoparticles transport experiments.

模拟人血脑屏障的三重培养细胞系统

The delivery of drugs to the brain remains a challenge due to the blood-brain barrier's (BBB) highly specific and restrictive properties, which controls ...

生物学

An Innovative 3D-Printed Insert Designed to Enable Straightforward 2D and 3D Cell Cultures

The classical analyses of indirect communication between different cell types necessitate the use of conditioned media. Moreover, the production of conditioned media remains time-consuming and far from physiological and pathological conditions. Although a few models of co-culture are commercially available, they remain restricted to specific assays and are mostly for two types of cells. 
Here, 3D-printed inserts are used that are compatible with numerous functional assays. The insert allows the separation of one well of a 6-well plate into four compartments. A wide range of combinations can be set. Moreover, windows are designed in each wall of the compartments so that potential intercellular communication between every compartment is possible in the culture medium in a volume-dependent manner. For example, paracrine intercellular communication can be studied between four cell types in monolayer, in 3D (spheroids), or by combining both. In addition, a mix of different cell types can be seeded in the same compartment in 2D or 3D (organoids) format. The absence of a bottom in the 3D-printed inserts allows the usual culture conditions on the plate, possible coating on the plate containing the insert, and direct visualization by optical microscopy. The multiple compartments provide the possibility to collect different cell types independently or to use, in each compartment, different reagents for RNA or protein extraction. In this study, a detailed methodology is provided to use the new 3D-printed insert as a co-culture system. To demonstrate several capacities of this flexible and simple model, previously published functional assays of cell communication were performed in the new 3D-printed inserts and were demonstrated to be reproducible. The 3D-printed inserts and the conventional cell culture using conditioned media led to similar results. In conclusion, the 3D-printed insert is a simple device that can be adapted to numerous models of co-cultures with adherent cell types.

In this paper, a newly designed 3D-printed insert is presented as a model of co-culture and validated through the study of the paracrine intercellular communication between endothelial cells and keratinocytes.

创新的 3D 打印插入物，旨在实现简单的 2D 和 3D 细胞培养

The classical analyses of indirect communication between different cell types necessitate the use of conditioned media. Moreover, the production of ...