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 border="0" cellpadding="0" cellspacing="0" width="585">
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Growth Factor Reduced matrigel</td>
			<td style="width:83px;">BD biosciences</td>
			<td style="width:119px;">354230</td>
			<td style="width:167px;">Avoid repeat freeze thaw&#160;</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Lab-Tek glass hamber slides 8-well</td>
			<td style="width:83px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">177402</td>
			<td>Precool on ice&#160;</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Lab-Tek glass chamber slides 2- well&#160;</td>
			<td style="width:83px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">177380</td>
			<td>Precool on ice&#160;</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">goat anti mouse F(ab&#180;)2 fragment</td>
			<td style="width:83px;">Jackson Immunoresearch</td>
			<td style="width:119px;">115-006-006</td>
			<td></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Normal goat serum</td>
			<td style="width:83px;">Jackson Immunoresearch</td>
			<td style="width:119px;">005-000-121</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Fluorescent conjugated Alexa antibodies</td>
			<td style="width:83px;">Molecular probes</td>
			<td style="width:119px;"></td>
			<td>Please look at molecular probes website</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Prolong gold antifade reagent with DAPI</td>
			<td style="width:83px;">Molecular Probes</td>
			<td style="width:119px;">P36935</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">3D Culture Cell Harvesting kit</td>
			<td style="width:83px;">Trevigen</td>
			<td style="width:119px;">3448-020-k</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-Apha-6-integrin</td>
			<td style="width:83px;">Fisher Scientifics&#160;</td>
			<td style="width:119px;">MAB1378</td>
			<td>1:00 dilution for IF</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-GM130</td>
			<td style="width:83px;">BD Biosciences</td>
			<td style="width:119px;">610822</td>
			<td>1:00 dilution for IF</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-phospho-histone 3 (Ser10)FITC Conjugated</td>
			<td style="width:83px;">Fisher Scientifics&#160;</td>
			<td style="width:119px;">16-222</td>
			<td>1:100 dilution for IF</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-Cleaved caspase 3 (Asp 175)</td>
			<td style="width:83px;">Cell Signaling</td>
			<td style="width:119px;">9661s</td>
			<td>1:50 dilution for IF</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-E-cadherin</td>
			<td style="width:83px;">BD Biosciences</td>
			<td style="width:119px;">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

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

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

人类神经内分泌肿瘤细胞株为研究人类神经内分泌肿瘤治疗的三维模型

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

科研

医学

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 ...

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 ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

注射原位乳腺癌细胞到小鼠乳腺脂肪垫来研究肿瘤的生长。

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

使用腺相关病毒作为一种工具来研究疾病中的视网膜障碍

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

三维（3D）肿瘤球体侵袭试验

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

软琼脂集落形成实验利用识别的致瘤性乳腺癌细胞抑制剂

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

人垂体腺瘤细胞的三维海藻酸钠珠文化

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

使用Saccadometry与深部脑刺激来研究正常和病理脑功能

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...

A Clinical Trial Assessing the Safety, Efficacy, and Delivery of Olive-Oil-Based Three-Chamber Bags for Parenteral Nutrition

Limited evidence exists to precisely estimate efficacy and safety differences between parenteral nutrition (PN) prepared using olive-oil-based three-chamber bags (3CBs) and soybean-oil-based compounded bags (CoBs) in hospitalized adult patients. We designed a multicenter, randomized, prospective, open-label, noninferiority protocol to compare the efficacy, safety, and distribution of olive-oil-based 3CBs and soybean-oil-based CoB formulations in adult Chinese patients requiring PN during surgical intervention. Subjects were randomized to receive either one of the study treatments using an interactive voice or web-based recognition system in accordance with the randomization code. Randomization was further stratified based on the study site and surgical category. Both treatment groups received similar amounts of calories and protein. In addition, the two study treatments contained a similar composition of the amino-acid component. The only difference between the two PN formulations was the lipid constitution. The duration of administration of study treatments was a minimum of 5 days up to a maximum of 14 days after the surgical procedure. The primary efficacy endpoint was serum prealbumin levels on day 5 of the study. Noninferiority was proved if the anti-log of the lower bound of the 95% confidence interval (CI) of the treatment difference was at least 0.80. Other efficacy measures included treatment preparation time; duration to achieve tolerability of oral nutrition; associated infectious complications; length of hospitalization; and laboratory assessment of markers of nutrition, inflammation, metabolism, and oxidative stress. A total of 458 patients were enrolled in the study. The results showed that olive-oil-based 3CBs were non-inferior to soybean-based CoBs, besides being well tolerated. The infection rate was found to be significantly lower in the olive-oil-based 3CB group. Thus, this study may be used as a reference for future research on lipid emulsion and 3CBs.

Here, we present a protocol for a study comparing the efficacy, safety, and delivery of olive-oil-based 3CB and soybean-oil-based CoB formulations in adults requiring parenteral nutrition. The results revealed that olive-oil-based 3CBs is non-inferior and well tolerated compared to soybean formulations.