这项工作是由美国国立卫生研究院资助R01 CA107469，R01 CA125577和U01CA154224到CG Kleer的，密西根大学的癌症中心支持大学的支持。

材料1。制备及文化传媒<ol><li>解冻生长因子降低（GFR）的基底膜，在4°CO / N。 </li><li>热身完整的媒体所需的细胞系在37°C。 MCF10A-ATCC指定的介质; HME-Ham氏F-12培养基补充有5％胎牛血清，1微克/毫升氢化可的松，5微克/毫升的胰岛素，10毫微克/毫升的表皮生长因子，和100纳克/毫升的霍乱毒素; SUM149-Ham氏F-12介质补充有5％FBS，2微克/毫升氢化可的松和5微克/毫升胰岛素;和MDA-MB-231-10％FBS-DMEM中</li><li>预冷冰上的8孔腔滑。 </li><li>准备组织培养罩和用品。 </li></ol> 2，三以及8商会幻灯片立体养殖<ol><li>外套各孔在8孔滑动用40微升的GFR基质胶的。添加基质胶中的幻灯片的中心，然后用P200移液管尖端传播。尽量分散尽可能均匀，避免气泡和过扩展可导致半月板形成。 </li><li>孵育的幻灯片，在37℃5％CO 2条件下。而基质胶的固化，胰蛋白酶消化细胞，收集并在组织培养中的离心旋转，在150×g离心4分钟。 </li><li>计数细胞并稀释至12,500个细胞/ ml在相应细胞系的完整的媒体。加GFR的Matrigel至2％，并添加400微升到基质胶预涂腔滑动的各孔中。 </li><li>孵化中的CO 2培养箱的幻灯片。给新鲜完整的媒体的变化，每4 个天，直到15天。 </li><li> 治疗药物抑制剂:对于抑制剂的研究添加小分子抑制剂的完整的媒体在电镀后辅以抑制剂新鲜媒体的变化的时候，每三天，而生长在基质胶的GFR的细胞。 </li><li> 治疗用纯化的蛋白: <ol><li>板上的细胞以下步骤2.1-2.3。让文化来生长4天，随后血清starvati上16小时。 </li><li>在第5天，治疗的细胞与纯化的蛋白质的适当的浓度在0.1％FBS补充培养基中的血清饥饿的细胞。术后第2天给予新鲜的媒体变化与纯化蛋白质。 </li><li>在10日与完整的HME介质更换条件培养基。让文化成长为另外5天。 </li></ol></li><li> 生长在基质胶腺泡结构的免疫荧光染色: <ol><li>制备2％多聚甲醛，0.5％的Triton X-100和100mM甘氨酸中的1×PBS，pH为7.4。制备免疫缓冲液（IF缓冲液），其包含的1×PBS中含有0.1％牛血清白蛋白，0.2％的Triton X-100，0.05％Tween-20的。 </li><li>使用P200的枪头小心吸的条件培养基。 </li><li>修复腺泡结构，用2％在室温（RT）新鲜制备的多聚甲醛固定20分钟。 </li><li>透化的细胞用0.5％Triton X-100的10分钟，在4℃下</li><li>洗净文化三大次100mM甘氨酸，10〜15分钟，每次洗涤。 </li><li>在中频缓冲器10％山羊血清，称为主块，1小时，在室温下阻断细胞。 </li><li>孵育用20微克/毫升的山羊抗小鼠的F（ab&#39;）2片段在30分钟主块缓冲器中的培养物。这个步骤是重要的，以阻止免疫小鼠IgG种类在基质胶。 </li><li>与一抗孵育第二封闭液（小学块20μg/ ml的羊抗鼠的F（ab&#39;）2片段）O / N在4°C。 </li><li>与中频缓冲器为每一个温和的摇摆10-15分钟，洗三次。 </li><li>孵育45分钟，在中频缓冲器荧光标记的二抗。 </li><li>洗3次，每次10 min与中频缓冲器与温和的摇摆。 </li><li>安装滑轨含DAPI以可视化的核防褪色剂。 </li><li>让片干燥O / N在室温。 </li><li>图像采集:采集共聚焦图像的殖民地英里dsection生长在基质胶的顶部细胞。欲了解更多信息，取得了一系列的光学部分整个被Z堆叠腺泡结构的整个长度。 </li></ol></li></ol> 3，三维立体培养中2井室幻灯片的免疫印迹<ol><li>预冷冰2井室幻灯片。按照步骤2.1-2.4扩大试剂为2孔腔滑如大衣，160微升基底膜，并添加1.6毫升细胞/基质胶混合到2孔滑动的每一个孔。 </li><li>收获的基质胶使用市售的三维培养细胞收集试剂盒，按照制造商的说明腺泡结构。 </li><li>稀释细胞清洗和细胞收集缓冲区为1x和预冷至4℃。洗涤基质胶和撞出应进行冰上。 </li><li>洗腺泡结构3X使用的细胞洗涤缓冲液。 </li><li>用细胞收集BU收集腺泡到15ml离心管中FFER随后孵育30分钟上的摇杆在4℃下混合悬浮液以及用1毫升移液管。这将破坏基质胶中的小块，并释放大部分细胞在细胞收获缓冲液中。 </li><li>颗粒细胞在200 XG在文化离心机。从沉淀的细胞用P200移液管尖取出水凝胶的漂浮层。 </li><li>重悬随试剂盒提供在SDS样品缓冲液中裂解。的RIPA裂解缓冲液中也可使用。 </li></ol>恶性与良性乳腺表现型4。定量<ol><li>以下步骤2.1-2.4生长的细胞以一式三份对各治疗组。 </li><li>采取腺泡结构的相位对比图像从每个孔在5X或使用连接到滑动移位器和一个计算机相衬显微镜10X分辨率。从一帧幻灯片移动到另一个，从而覆盖腔滑动的整个井所拍摄的图像。 </li><li>计数腺泡结构的总数，单轮平腺泡和multiacinar结构或缺乏组织胡乱成长结构的数量以及与分支过程从它发出的数量。 </li><li>计算单轮平腺泡结构与恶性结构的比例和情节的柱状图。通过学生t-检验计算显着性。 </li></ol>

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作者宣称没有竞争的财务权益。

在这里，我们已经描述了乳腺上皮细胞对重组基底膜的三维立体培养和本系统恶性与良性乳腺表现型来区分的用处。此系统还可以用于从其他腺体组织培养不同类型的细胞和已报道已被成功地用于培养的前列腺上皮细胞，支气管上皮细胞和甲状腺上皮细胞，仅举几例12-14。三维培养的重要性在于，它是生理学相关的模型。三维培养提供了深入了解，导致腺体结构破坏的恶性转化过程中的分...

三维上重建基底膜乳腺上皮细胞培养物（三维）是一种重要的模型系统来研究复杂的表型和正常乳腺和乳腺癌2,3的相应的信令。乳房的功能单元是终末导管小叶单元（TDLU），它由一个小导管，其分支成腺泡。腺泡是高度组织的结构中，两个层细胞，上皮细胞和肌上皮细胞，这是由基底膜5包围的组成。正常的腺泡的最显着特征是细胞极化，附着在底层基底膜和专门的细胞与细胞的接触。这个复杂的组织被破坏的浸润性癌。被广泛使用的二维培养，其中细胞生长为单层，不允许腺泡的形成。因此，该单层培养缺乏提供一个系统来捕捉上皮细胞之间的错综复杂的关系在正常乳腺和乳腺癌放松管制。乳腺癌细胞的功能性的单种培养上皮已经开发了几个实验室2,3。三维培养涉及乳腺癌细胞在基质胶，这是从Engelbreth-的Holm-群小鼠肉瘤细胞衍生的溶解提取物，并且是可商购的生长。我也喜欢用其他的胶原基质的3D。乳腺细胞可以通过3D嵌入式测定法，其中细胞培养嵌在基质胶2中培养在三维。此外，细胞可以通过3D顶部化验培养，也被称为3D叠加分析，这是符合成本效益，因为它需要的基质胶量较小，并促进集落形成时间推移监测相衬成像，是理想的原位成像2 -3。在上面试验中的三维已将用于定义腺泡表型和基因表达概况7之间的相关性。 三维培养可effectiv伊利用于从浸润癌区分正常和良性细胞。正常和良性乳腺细胞形成生长停止极化结构，在中心一个明确的管腔上，而基底膜浸润性癌细胞生长力强和随意，没有结算管腔。 E6/E7永生化人乳腺上皮细胞和MCF10A细胞系，这是一个自发永生化细胞系来自一个36岁的患者，纤维囊性变孤立的，已成功地用于夺回乳腺良性表型3D 3,8，并担任一个模型系统来研究几个分子8,9的致癌和抑癌功能。这些良性细胞可以被工程化以引入慢病毒/逆转录病毒操纵感兴趣的基因（次）。如果感兴趣的基因（s）是一种肿瘤抑制基因，shRNA的介导的敲除会导致，而恶性转化，如果感兴趣的基因是良性细胞癌基因的过度表达应该表现出恶性表型。在这两种情况下形成的三维腺泡结构可以捕获恶变。 良性的单细胞接种于3D开始增生并形成圆球形结构。 5-8天的细胞这个球形集合体将分化为细胞的周围偏振层，其与所述基质胶接触，并减少极化的细胞，缺乏与基质胶接触的内质。在接下来的10-15天的内细胞将开始凋亡形成一个清晰的管腔死。恶性肿瘤细胞将增长胡乱失去极性。高度恶性细胞通常形成分支结构。这些差异在表型可以通过时间的推移相衬成像和原位与相关分子标记免疫染色被捕获。最常用的标记是α-6-整，基底极性的标记，与所述增殖标志物磷酸化组蛋白3和apoptot组合IC标记物裂解的caspase-3。后者是重要的，以确定是否有管腔形成在中央的腺泡，正常和良性乳腺细胞的特征。其他极性和细胞间接触标志物包括GM130，层粘连蛋白，企业风险管理，E-cadherin的。交替的乳腺癌细胞系可被工程化来操纵感兴趣的基因。在这种情况下，复归到一个更大的增长被捕良性的表型可以被用作一个读出从癌症的表型区分。 三维培养也可以仔细用来划定参与恶性转化10-11的信号通路。使用上面提到的读数，药理抑制剂，阻断性抗体或重组蛋白可用于在分子信号传导，导致恶变被精确定位。与来自不同实验室的不断努力，可以从三维提取细胞，提取RNA，并制备裂解物用于免疫印迹和免疫noprecipitation。这些策略在协议中，并逐步详细地描述。

<table><tbody><tr><td>Growth Factor Reduced Matrigel</td><td>BD Biosciences</td><td>354230</td><td>Avoid repeat freeze thaw&amp;nbsp;</td></tr><tr><td>Lab-Tek glass hamber slides 8-well</td><td>Thermo Fisher Scientific</td><td>177402</td><td>Precool on ice&amp;nbsp;</td></tr><tr><td>Lab-Tek glass chamber slides 2-well&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>177380</td><td>Precool on ice&amp;nbsp;</td></tr><tr><td>Goat anti mouse F(ab&amp;acute;)&lt;sub&gt;2 &lt;/sub&gt;fragment</td><td>Jackson Immunoresearch</td><td>115-006-006</td><td></td></tr><tr><td>Normal goat serum</td><td>Jackson Immunoresearch</td><td>005-000-121</td><td></td></tr><tr><td>Fluorescent conjugated Alexa antibodies</td><td>Molecular Probes</td><td></td><td>Please look at molecular probes website</td></tr><tr><td>Prolong gold antifade reagent with DAPI</td><td>Molecular Probes</td><td>P36935</td><td></td></tr><tr><td>3D Culture Cell Harvesting kit</td><td>Trevigen</td><td>3448-020-k</td><td></td></tr><tr><td>Anti-Apha-6-integrin</td><td>Fisher Scientific</td><td>MAB1378</td><td>1:100 dilution for IF</td></tr><tr><td>Anti-GM130</td><td>BD Biosciences</td><td>610822</td><td>1:100 dilution for IF</td></tr><tr><td>Anti-phospho-histone 3 (Ser10)FITC Conjugated</td><td>Fisher Scientific</td><td>16-222</td><td>1:100 dilution for IF</td></tr><tr><td>Anti-Cleaved caspase 3 (Asp 175)</td><td>Cell Signaling</td><td>9661s</td><td>1:50 dilution for IF</td></tr><tr><td>Anti-E-cadherin</td><td>BD Biosciences</td><td>610181</td><td>1:200 dilution for IF</td></tr></tbody></table>

three dimensional cultures: a tool to study normal acinar architecture vs. malignant transformation of breast cells

乳腺浸润性癌是一组恶性上皮性​​肿瘤特征的侵袭邻近组织，并倾向转移的。信号癌细胞和微环境之间的相互作用发挥对乳腺癌的生长和生物学行为1强大的影响力。然而，大多数来自细胞外基质，这些信号的丢失或当细胞生长在二维培养（2D）作为单层的相关性被充分研究。近年来，三维（3D）上的重组基底膜培养已成为首选的方法扼要重述良性和恶性乳腺细胞的组织结构。在三维培养的细胞保留来自细胞外基质的重要线索，并提供一个生理相关的体外系统2,3。值得注意的是，有越来越多的证据表明细胞有不同的行为时，在3D种植相比，2D 4。三维培养可以有效地使用，以从良性乳腺表型分化的恶性表型的装置和用于支撑的细胞和分子信号传导参与3。一种良性上皮细胞的显着特点是它们被极化，使得根尖细胞质是朝向管腔和基底细胞质搁置在基底膜。这apico - 基底极性消失在乳腺浸润性癌，其特点是细胞解体和形成网状和分支小管是随意渗入周围的基质。良性腺体和浸润癌之间的这些病理的差异可以被复制在3D 6,7。使用了适当的读奏像单轮腺泡结构的定量，或差的验证分子标记物的细胞增殖，极性和凋亡与其它分子和细胞生物学技术，三维文化视角组合表达E能提供更好的理解过程中的恶性转化，并划定了负责信号的细胞变化的重要工具。

三维培养物可被有效地用于区分人类乳腺上皮细胞的恶性表型的良性乳腺。镀在基质胶单良性细胞成长为有组织的球形结构采用X射线相衬成像，可以很容易地成像为单轮平腺泡在一个平面上。镀在基质胶的单一的恶性细胞生长随意，显示出非常高的折射率，而且难以像在一个平面上。 如图1，良性HME和MCF10A细胞形成球形寻找腺泡而高转移性乳腺癌细胞系SUM149和MDA-MB-231成长为分支的管?...

Watch this Scientific Journal Video about Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells at JoVE.com

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

对重组基底膜乳腺上皮细胞的三维培养是扼要重述在体内的体系结构的良性乳房，并从良性乳腺表型分化的恶性表型的有效方法。重要的是，该系统可以应用到研究浸润性癌中的其他组织中。

三维文化:一种工具来研究正常腺泡结构与恶性转化的乳腺细胞

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

three-dimensional-cultures-tool-to-study-normal-acinar-architecture

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

19.8K 次观看.  University of Michigan Comprehensive Cancer Center. 对重组基底膜乳腺上皮细胞的三维培养是扼要重述在体内的体系结构的良性乳房，并从良性乳腺表型分化的恶性表型的有效方法。重要的是，该系统可以应用到研究浸润性癌中的其他组织中。 

视频: Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract (ECME)-based Three-dimensional System

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, thus greatly influencing normal tissue homeostasis and disease outcome. In breast cancer, tumor-associated macrophages (TAMs) play a critical role in disease progression, metastasis, and recurrence; therefore, understanding the mechanisms of monocyte chemoattraction to the tumor microenvironment and their interactions with tumor cells is important to control the disease. Here, we provide a detailed description of a three-dimensional (3D) co-culture system of human breast cancer (BrC) cells and human monocytes. BrC cells produced high basal levels of regulated on-activation, normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1), and granulocyte-macrophage colony-stimulating factor (G-CSF), while in co-culture with monocytes, pro-inflammatory cytokines Interleukin (IL)-1 beta (IL-1&#946;) and IL-8 were enriched together with matrix metalloproteinases (MMP)-1, MMP-2, and MMP-10. This tumor stroma microenvironment promoted resistance to anoikis in MCF-10A 3D acini-like structures, chemoattraction of monocytes, and invasion of aggressive BrC cells. The protocols presented here provide an affordable alternative to study intra-tumor communication and are an example of the great potential that in vitro 3D cell systems provide to interrogate specific features of tumor biology related to tumor aggression.

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ECME-based Three-dimensional System

Here, we describe a three-dimensional culture method to analyze the morphology of primary breast cancer cells, as well as to study their direct/indirect interactions with monocytes and the outcomes such as collagen degradation, immune cell recruitment, cell invasion, and promotion of cancer-related inflammation.

基于细胞外基质提取物 (ECME) 的三维系统中单核细胞与原代乳癌细胞的通讯分析

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, ...

科研

癌症研究

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

球体的检测，以测量TGF -β诱导的入侵

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

医学

Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture

The differentiation of acinar cells to ductal cells during pancreatitis and in the early development of pancreatic cancer is a key process that requires further study. To understand the mechanisms regulating acinar-to-ductal metaplasia (ADM), ex vivo 3D culture and differentiation of primary acinar cells to ductal cells offers many advantages over other systems. With the technique herein, modulation of protein expression is simple and quick, requiring only one day to isolate, stimulate or virally infect, and begin culturing primary acinar cells to investigate the ADM process. In contrast to using basement membrane matrix, the seeding of acinar cell clusters in collagen I extracellular matrix, allows acinar cells to retain their acinar identity before manipulation. This is vital when testing the contribution of various components to the induction of ADM. Not only are the effects of cytokines or other ectopically administered factors testable through this technique, but the contribution of common mutations, increased protein expression, or knockdown of protein expression is testable via viral infection of primary acinar cells, using adenoviral or lentiviral vectors. Moreover, cells can be re-isolated from collagen or basement membrane matrix at the endpoint and analyzed for protein expression.

Primary acinar cell isolation, protein expression or activity modulation, culture, and down-stream applications are abundantly useful in the ex vivo study of acinar-to-ductal metaplasia (ADM), an early event in the development of pancreatic cancer.

三维细胞培养中胰腺肌囊内增生的模拟与操纵

The differentiation of acinar cells to ductal cells during pancreatitis and in the early development of pancreatic cancer is a key process that requires ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

人类乳腺上皮细胞致癌转化的体外评价

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

三维组织培养模型研究人类原发性骨髓和恶性肿瘤

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Phospholipid Mediator Induced Transformation in Three-Dimensional Cultures

Several models have been developed to study cancer, such as rodent models and established cell lines. Valuable insights into carcinogenesis have been provided by studies using these models. Cell lines have provided an understanding of the deregulation of molecular signaling associated with breast tumorigenesis, while rodent models are widely used to study cellular and molecular characteristics of breast cancer in vivo. The establishment of 3D cultures of breast epithelial and cancerous cells aids in bridging the gap between in vivo and in vitro models by mimicking the in vivo conditions in vitro. This model can be used to understand the deregulation of complex molecular signaling events and the cellular characteristics during breast carcinogenesis. Here, a 3D culture system is modified to study a phospholipid mediator-induced (Platelet Activating Factor, PAF) transformation. Immunomodulators and other secreted molecules play a major role in tumor initiation and progression in the breast. In the present study, 3D acinar cultures of breast epithelial cells are exposed to PAF exhibited transformation characteristics such as loss of polarity and altered cellular characteristics. This 3D culture system will assist in shedding light on genetic and/or epigenetic perturbations induced by various small molecule entities in the tumor microenvironment. Additionally, this system will also provide a platform for the identification of novel as well as known genes that may be involved in the process of transformation.

The present protocol describes the setting up of 3D 'on top' cultures of a non-transformed breast epithelial cell line, MCF10A, that has been modified to study Platelet Activating Factor (PAF) induced transformation. Immuno-fluorescence has been used to assess the transformation and is discussed in detail.

磷脂介质诱导的三维培养物转化

Several models have been developed to study cancer, such as rodent models and established cell lines. Valuable insights into carcinogenesis have been ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

乳腺癌细胞侵袭定量使用三维（3D）模型

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, including breast carcinomas, are complex 3D tissues composed of cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix (ECM). Yet most in vitro models of breast carcinoma consist only of cancer epithelial cells, omitting the stroma and, therefore, the 3D architecture of a tumor in vivo. Appropriate 3D modeling of carcinoma is important for accurate understanding of tumor biology, behavior, and response to therapy. However, the duration of culture and volume of 3D models is limited by the availability of oxygen and nutrients within the culture. Herein, we demonstrate a method in which breast carcinoma epithelial cells and stromal fibroblasts are incorporated into ECM to generate a 3D breast cancer surrogate that includes stroma and can be cultured as a solid 3D structure or by using a perfusion bioreactor system to deliver oxygen and nutrients. Following setup and an initial growth period, surrogates can be used for preclinical drug testing. Alternatively, the cellular and matrix components of the surrogate can be modified to address a variety of biological questions. After culture, surrogates are fixed and processed to paraffin, in a manner similar to the handling of clinical breast carcinoma specimens, for evaluation of parameters of interest. The evaluation of one such parameter, the density of cells present, is explained, where ImageJ and CellProfiler image analysis software systems are applied to photomicrographs of histologic sections of surrogates to quantify the number of nucleated cells per area. This can be used as an indicator of the change in cell number over time or the change in cell number resulting from varying growth conditions and treatments.

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained.

制备及分析<em&gt;体外</em&gt;三维乳腺癌代理项

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, ...

生物工程

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

通过微分试穿素生成马赛克乳腺有机体

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

发育生物学

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

从三维混合细胞球体共培养的乳腺上皮细胞的隔离

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...