Name: imaging- and flow cytometry-based analysis of cell position and the cell cycle in 3d melanoma spheroids
Uploaded: 2015-12-28T14:00:00.000+00:00
Duration: 10 min 44 s
Description: Watch this Scientific Journal Video about Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids at JoVE.com

We thank Ms. Danae Sharp and Ms. Sheena Daignault for technical assistance. We thank Dr. Atsushi Miyawaki, RIKEN, Wako-city, Japan, for providing the FUCCI constructs, Dr. Meenhard Herlyn and Ms. Patricia Brafford, The Wistar Institute, Philadelphia, for providing cell lines, the Imaging and Flow Cytometry Facility at the Centenary Institute for outstanding technical support. We thank Mr. Chris Johnson and Dr. Andrew Barlow for Volocity software technical support. N.K.H. is a Cameron fellow of the Melanoma and Skin Cancer Research Institute, Australia. K.A.B. is a fellow of the Cancer Institute New South Wales (13/ECF/1-39). W.W. is a fellow of the Cancer Institute New South Wales (11/CDF/3-39). This work was supported by project grants RG 09-08 and RG 13-06 (Cancer Council New South Wales), 570778 and 1051996 (Priority-driven collaborative cancer research scheme/Cancer Australia/Cure Cancer Australia Foundation), 08/RFG/1-27 (Cancer Institute New South Wales), and APP1003637 and APP1084893 (National Health and Medical Research Council).

1. FUCCI转导和细胞培养<ol><li> FUCCI转导<ol><li>创建细胞系稳定表达FUCCI使用慢病毒共转导如前所述7构造mKO2-hCdt1（30-120）和MAG-hGem（1-100）15。 注:FUCCI系统现在市售的。 </li><li>生成子的克隆与明亮的荧光由单细胞分选。排序单阳性细胞都AG和KO（黄色）通过荧光激活细胞分选入96孔板如前所述7,16。 </li></ol></li><li>黑色素细胞培植<ol><li>培养C8161人黑素瘤细胞如前所述7,17。 </li></ol></li></ol> 2.三维球体的形成（如前所述3,9） <ol><li>琼脂糖板的准备<ol><li>溶解1.5％琼脂糖（或稀有琼脂）在100毫升的Hank氏平衡盐溶液（HBSS）或磷酸盐buffereð盐水（PBS）通过在微波中3-5分钟旋流沸腾。确保琼脂糖完全溶解。 </li><li>立即用多通道移液器分配每孔100μl的琼脂糖溶液至平底96孔组织培养板。 注意:琼脂糖可以以提示固化后的短时间内，为了克服这个问题，必须提示板之间改变，如果使多个琼脂糖平板。 </li><li>留在平坦表面上96孔板硬化琼脂糖至少1小时。 </li></ol></li><li>电池的制备和覆盖到琼脂糖<ol><li>生长细胞到约80％汇合的T75烧瓶。用10毫升HBSS清洗。 </li><li>用1.5毫升的0.05％胰蛋白酶/乙二胺四乙酸（EDTA），重悬在10ml正常培养基分离细胞。 </li><li>计数使用血球18或其他自动计数法的细胞。 </li><li>重悬的细胞以在范数25,000个细胞/ ml的终浓度人网上平台。 </li><li>覆盖琼脂糖孔用200μl细胞悬浮液为每孔5,000个细胞的终数目。 </li><li>回位盘到细胞培养孵化器（5％CO 2和37℃），以形成球状体在约3天。 注意:采取以形成球体的时间不同的细胞系之间变化。某些细胞系不形成使用该方法在所有的球状体。 </li><li>图像球体形态与使用4X物镜和相衬滤波器倒置显微镜。的细胞系，成功地形成球状体将产生紧凑，大致球状细胞聚集，并且应该有每口井只有一个球体。 注意:可选:一旦球体形成，它们可以被移植到胶原或其他基质的生长及侵袭3,7,17 ​​的进一步分析。从胶原除去球体，治疗用500μl的2毫克/毫升胶原酶在37℃下，直至胶原溶解足够的spher的OID来自由入培养基（约30分钟）。轻轻地取出用1毫升吸管球体。 </li><li>用于切片/成像修复球状体（无论是分离的由胶原或生长3-5天在琼脂糖）在10％的中性缓冲的福尔马林（小心:福尔马林应在通风橱中使用）至少2小时，在室温（RT） ，或隔夜（O / N）在4℃。球状​​体可以被存储在HBSS在4℃下数天。 </li></ol></li></ol> 3.球体Vibratome切片<ol><li>溶解5克在100ml的HBSS低熔点琼脂糖在500毫升玻璃瓶中微波炉。使用带有旋转介质热定型。注意:保持盖子松动时沸腾的溶液中，使得该压力可以被释放。使用防护手套，防热处理热玻璃奶瓶时要保护手烫伤。 </li><li>等到琼脂糖稍微冷却 - 这样的瓶子可以不烧手触摸。 </li><li>倒approximately 2毫升琼脂糖成Coulter计数器杯或类似塑料模具的底部。立即吸固定球体（见2.2.8）中的液体可能的最小体积使用1ml吸管。加入球体的琼脂糖。几个球体（最多10个）可以每杯加入。 </li><li>覆盖球体用2毫升更多的琼脂糖。为此快，从而避免琼脂糖硬化的第二层中加入之前。 </li><li>允许琼脂糖在RT在暗处硬化，至少3小时。在这一点上的杯子可被存储的时间很短，在4℃用2-3毫升的HBSS覆盖，以防止脱水。 </li><li>从杯子琼脂糖删除。修剪含有球状体，以便它装配在vibratome金属块，和球状体接近琼脂糖顶部的琼脂糖。球状​​体可以被看作是在琼脂糖内小白物体。强力胶琼脂糖块。 </li><li>装满蒸馏水vibratome并安装在vibratome块。摆好vibratome削减100微米的部分使用低转速（设定2）和高振动（设置8）。设置切刃角至16°。转vibratome上，打开vibratome光并切断从琼脂糖的顶部部分直到100微米的球体部分被切断。将切球体部分为HBSS的24孔板。 </li><li>选择中间部分的图像分析。山路段上的幻灯片被转移部分平板上使用镊子滑动。填有10毫升注射器筒与真空油脂;为了防止在安装介质从流失滑动的边缘，并且帮助密封盖玻片下的部分添加的细线的周围部的边缘真空润滑脂。覆盖部分与封固剂和盖玻片。密封盖玻片指甲油的边缘。幻灯片保存在4℃前成像。 </li></ol> 4.共聚焦图像采集<ol><li>走中间FUCCI球体部分的使用10X的Z切片图像或20X物镜如前所述7。确保没有重叠或串扰Kusabira Orange2和Azami绿色之间发生。如果比较多个球体部分的图像分析，保持激光功率和其他设置的样品之间的相同。 </li></ol> 5.赫斯特染料扩散分析的流量排序<ol><li>转移活球状体（琼脂糖播种后3-5天），以15毫升管。让球体重力沉降到管底部。几个球体可以每管被染色。需要大约20球状体来获得足够的细胞数量用于流式细胞术。 </li><li>删除多余的介质，并加入1毫升10μM赫斯特稀释在普通培养基上。孵育球体在37℃下约1小时。用温柔轻拂孵化过程中一次或两次，悬浮球体。 注意:培养时间可以变化，以改变多远的Hoechst染料渗入球体。这将有所不同的基础上球体的尺寸，密度和细胞系。对于一些大型/致密的球体可以有最大的染料渗透小于100％。 </li><li>用5毫升的HBSS利用重力沉降轻轻冲洗球体。 <ol><li>可选:对于染料渗透成像:在室温或O / N固定球体用10％中性缓冲福尔马林至少2小时，在4℃。制备vibratome部分，如在步骤3和4如上所述安装在滑动和图像上的共焦。 </li></ol></li><li>至在37℃下制备的细胞进行流式细胞术，trypsinize球状体用1ml 0.05％胰蛋白酶/ EDTA约30分钟，轻弹每10分钟，打破球体分开。 注意:球状体是很难得到单细胞悬液，条件可能需要优化。这一过程后C8161 4日龄的球状体生存能力约95％。 </li><li>染色细胞根据制造商的协议的近红外活/死染色。用HBSS洗涤，然后用1修复毫升10％中性福尔马林缓冲液大约30分钟。细胞可被存储在HBSS在4℃下进行流分析前几天。 </li></ol>赫斯特彩绘FUCCI球体的6流式细胞仪分析<ol><li>确保单元，不以通过细胞过滤网来成群在一起。重悬的Hoechst以1-5×10 6个细胞/ ml的浓度染色FUCCI球状体在冰冷的FACS洗涤（PBS中的5％胎牛血清）。转移200μl的样品，以5毫升圆底管中。 </li><li>准备以下单一颜色对照样品中6.1描述:未染色的黑色素细胞;贴壁黑色素细胞染色用10微米赫斯特1小时; Azami绿色只表达黑色素瘤细胞;和Kusabira橙色仅表达黑色素瘤细胞。 注:单色控制可固定在福尔马林中并存储在FACS有0.1％的叠氮化钠洗在4℃数周甚至数月。 </li><li>运行单细胞悬液Ø呐流式细胞分析仪适当的激光和单色/未染色的控制如前所述7,16。 </li><li>使用商业软件，流式细胞仪等的FlowJo如下分析内部与外部球体细胞: <ol><li>通过门控的主要细胞群的前向散射光（FSC）与侧散射光（SSC）清除杂物。 </li><li>通过门控使用FSC（区）与FSC（高）的单细胞移除双峰</li><li>门的活细胞通过门控上的活/死了较低的人口。 </li><li>限定Hoechst的高细胞，根据来自用Hoechst贴壁细胞的阳性信号选通，为"外"的细胞。 </li><li>定义Hoechst的低或阴性细胞，根据来自未染色的控制信号选通，为"内部"的细胞。 </li><li>选通内外种群后，细胞可进一步选通为FUCCI红色（G1），黄色（S早期），绿（S / G2 / M）或负（早期G1）。 注:C可能需要使用单一的色彩控制ompensation正确定义FUCCI红色，黄色，绿色和阴性群。 </li></ol></li><li>一旦测定了优化和通过共焦显微镜Hoechst的渗透验证，而不固定在流动细胞分选，如前所述7,16为活的细胞亚群的进一步分析运行的细胞。 </li></ol> FUCCI球体部分7.图像分析<ol><li>打开软件如 。 Volocity，选择定量唯一的配置。 </li><li>创建新库。从共聚焦拖动原始数据文件到库导入FUCCI球体共聚焦图像文件到软件中。或者，使用文件&gt;导入命令。 </li><li>去测量标签来建立一个图像分析协议。该协议是通过拖放以正确的顺序相应的命令在协议如下窗口建设。 </li><li>查找OBJ学分的绿色通道:拖动找对象命令（在&#39;查找&#39;）复制到协议窗口中，选择正确的通道，在找对象protocol.Add一个开放的命令（在&#39;加工&#39;中），然后一个单独的触摸物体命令（在"加工"发现 - 这将分离细胞）。排除由粒径&lt;50微米的对象（在"过滤"发现 - 这将排除小非细胞对象）。 注意:变量如查找对象的阈值，开放迭代次数，对象的大小要分离和对象的大小，以排除必须手动优化，直到对象掩码匹配图像。 </li><li>发现在红色通道对象:重复如上的红色通道找对象。绿色和红色协议可能需要单独的调整。 注:由于细胞核G2是，绿核往往稍大。 </li><li>确认发现是正确的对象:确认红色和绿色物体匹配红色和绿色的核的图像中通过关闭和绿色和红色通道（使用隐藏或显示信道命令），同时显示由所述协议发现红色和绿色口罩。反馈选项（尺寸&gt;反馈选项）可以用来修改对象口罩的外观。 </li><li>查找黄色的物体（早期S期细胞）:要找到黄色的单元格，添加一个相交红配绿细胞命令（在"合并"中）。排除按大小的物体（&lt;50微米，在"过滤"中）排除任何非小细胞对象。 </li><li>查找独家红色物体（G1期细胞）:要找到专门红核，减黄细胞的红细胞（在"合并"中）。按尺寸&lt;50微米，（在&#39;过滤&#39;中）排除对象删除创建的任何非小细胞对象。 </li><li>查找专门绿色物体（S / G2 / M期细胞）:重复的绿色通道找到专门的绿色。 没有德:如果仍有剩余的非细胞的目的，一个滤波器人口命令可以加入到异绿色或红色专属的协议（在"筛选"中）仅保留对象在具有形状因子大于0.25。这将删除非圆形物体。 </li><li>找到球体外形:查找使用查找球体轮廓对象命令中的绿色或红色通道（无论有更多的细胞周围的球体边缘）（在"查找"中）。甲低得多的阈值（在查找中找到的对象的变量）将需要被用来找到球体轮廓相比，个别细胞。 <ol><li>使用关闭命令加入对象（在&#39;加工&#39;中），然后填写对象孔（在&#39;加工&#39;中），然后用细过滤器（在"过滤"中）从对象去除噪声。最后，通过尺寸排除对象以除去较小的物体&lt;30000微米（根据不同的旋转椭球体的大小）。 注意:此协议将需要手动优化，直到球体轮廓是准确的。甲明图像，也可以使用 - 但是这通常是不太准确与球体部分，和一个反相命令（在&#39;结合&#39;找到）将需要被使用。 </li></ol></li><li>测量核的距离以球体外形:测量距离从黄色，完全绿色和红色的独家人口从小区重心的球体outline.To可视化的最小距离的边缘（在"有关&#39;中），这应该是从小区到最近的球体边缘，打开在反馈选项（尺寸&gt;反馈选项&gt;关系）的关系选项卡显示的距离。 </li><li>保存的协议。该协议可以被重新应用于使用测量其他图像&gt;恢复协议命令。 </li><li>创建一个测量项目（测量&gt;进行测量项目）。数据可以使用分析来分析功能。 <ol><li>转到在测量项目的分析选项卡。转到分析菜单（分析&gt;分析），分析从细胞质心（红色，绿色或黄色）的球体边缘，总结数量和人口组织的最小距离。 </li><li>计数在距边缘有一定距离中发现的细胞的数目创建一个过滤器（分析&gt;过滤器）。的过滤器在图2中最小距离是根据Hoechst的渗透（例如最小距离小于80给出了"外"人口）。另外，原始数据导出到电子表格进行进一步的分析。 </li></ol></li></ol>

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PerkinElmer contributed to the costs of publication.

半自动化图像分析，鉴定球体内G1被捕区域，和增殖外层。这种方法可以在活球状体用光学部分被使用，或在固定的球体部分，以识别变化不仅在细胞周期但标记表达（通过免疫荧光），细胞死亡，或细胞形态在这些不同的区域。不同球状体区域内的细胞运动性，也可以定量 - 如果连同一个细胞跟踪图像分析​​步骤的现场共焦时间推移成像溶液。关键的图像分析是，高品质的Z-切片的共聚焦图像获得更高分辨率的图像可以更好地识别细胞。一个限制与图像分析是，它是不可能正确找到每个细胞如果核是过于密集，或者如果在红色和绿色强度太大变化。 FUCCI阴性细胞（早期G1）不能够将与此图像分析方法，只有红（G1）发现，黄色（早S期）和绿色（S / G2 / M）。此处所描述的基于图像的分析方法的一个其它的缺点是，它不考虑球体的完整三维性质。虽然Volocity软件可以执行使用Z堆叠图像三维测量，共聚焦显微镜无法渗透和图像通过整个球体由于大尺寸。多光子成像可以使用，以获得Z堆叠整个球体的对三维测量7作为这种技术允许高达500微米的渗透，尽管有些球体可能仍然过大，无法通过此方法的图像整个球体。用于成像的整个球体的另一个选择是光片基显微镜，它允许从多个角度旋转椭圆体的可视化和穿透高达200微米的内部大球体20,21。成像策略的选择可通过球体大小，细胞堆积密度和细胞类型的影响。 赫司特染料diffusioN法用于通过流式细胞术在多细胞球状体分离内层和外层细胞于1982年22首先描述的，这种方法是基于这样的Hoechst的染料扩散慢慢进入球体，与外细胞被染色的第一这一事实。这里描述的协议结合赫斯特法与FUCCI 系统 15，这使得不仅分离内，外球体细胞，但也Hoechst的渗透可视相对于G1停滞细胞的内圈。这使得球体的G1被捕区域的精确分离。使用FUCCI单独不允许内的G1停滞细胞的从旋转椭球体的分离，因为该外增殖细胞还包含细胞G1期。一个限制是，该方法只允许球体的粗分离成两个区域（"外部"Hoechst的正和"内"Hoechst的负数）。该方法通过图像分析的好处，是使f小仪允许多个指标来一次测试，而活细胞进行后续分析，如基因或蛋白质的表达，重新培养在不同的环境条件下，药物治疗或其它分析的物理分离。 所述FUCCI球体系统在流式细胞仪和图像分析组合允许识别周围的坏死核心G1停滞细胞的内圈，和增殖细胞的外层。坏死和G1停滞在旋转椭圆体的中心发现的是，由于缺乏氧气和营养，以及发展为球体尺寸的成长。以前，我们和其他人已经表明共定位的pimonidazole缺氧标记与G1逮捕球体中心7,23的。我们还表明，增殖很大程度上发生在大约100微米，从旋转椭圆体的边缘。这球体12,23,24符合先前的研究，同时也与相关对distance 体内 25,26增殖来自最近的脉管细胞。 可用于分离基于它们在球体的位置进行流式细胞术或其它分析细胞的另一种方法是顺序胰蛋白酶24,27。在该方法中，球体的连续的"壳"是由在低温下短，顺序潜伏期用胰蛋白酶除去。然而，使用这种方法，它是更加难以确定外壳（以微米厚）的确切大小相比，Hoechst的染料扩散的方法，其中所述的Hoechst渗透可以直接可视化并测量被去除。 分离的"内部"和"外部"细胞染料扩散和/或图像分析可以扩展到小鼠FUCCI肿瘤异种移植研究。然而，对于细胞周期中的体内分析，脉管系统的存在也应た肯考虑在内，如细胞的肿瘤可能能够从脉管访问中心区域的氧和营养物质。

多细胞三维球状体已被被称为肿瘤模型数十年，但它只是在最近，他们已进入更常见的用法作为体外模型对许多实体癌症。它们越来越多地被用于高通量药物发现屏幕作为中间之间的复杂，昂贵和费时的体内模型和简单的，低成本的2D单层模型1-6。研究在2D培养往往无法在体内被复制。许多类型的癌症的球体模型是能够模仿的生长特性，药物敏感性，药物渗透，细胞-细胞相互作用，氧和营养物和坏死的发展是见于体内实体肿瘤6-11的有限的可及。球状体开发出坏死核心，包围芯静止或G 1被捕区域，并在球体7的周边增殖细胞。这些区域的发展可根据细胞密度，增殖率和球体12的尺寸而变化。据推测，见于这些不同子区的细胞异质性可能会导致癌症治疗电阻13,14。因此，分析细胞在这些区域中的能力分别是至关重要的理解肿瘤药物反应。 荧光泛素细胞周期指示符（FUCCI）系统是基于红色（Kusabira橙色-正）和绿色（Azami格林- AG）CDT1和geminin的荧光标记，这是降解的细胞周期15的不同阶段。因此细胞核出现红色于G1，黄S中的早期和绿色中的S / G2 / M期。我们在这里描述两个互补方法均使用FUCCI来识别细胞周期，随着使用成像软件或染料扩散流式细胞仪测定的，以确定细胞是否驻留在G1被捕中心或外细胞增生克环，和个体细胞从旋转椭圆体的边缘的距离。这些方法被开发在我们以前的出版物，在这里我们表明，黑色素瘤细胞在低氧区域中的旋转椭圆体的中心和/或在靶向治疗的存在下能够保持在G1期阻滞延长的时间段，并且可以重新在更有利的条件出现7进入细胞周期。

<table><tbody><tr><td>Hoechst 33342</td><td>Life Technologies</td><td>H3570</td><td></td></tr><tr><td>agarose low melting point</td><td>Life Technologies</td><td>16520-050</td><td>For sectioning</td></tr><tr><td>noble agar&amp;nbsp;</td><td>Sigma</td><td>A5431</td><td>For making spheroids</td></tr><tr><td>agarose for spheroids</td><td>Fisher Scientific</td><td>BP1356-100</td><td>For making spheroids</td></tr><tr><td>0.05% trypsin/EDTA</td><td>Life Technologies</td><td>25300-054</td><td></td></tr><tr><td>HBSS</td><td>Life Technologies</td><td>14175-103</td><td></td></tr><tr><td>10% formalin</td><td>Sigma</td><td>HT5014-1CS</td><td>CAUTION: Harmful, corrosive. Use Personal Protective Equipment, do &amp;nbsp;not breath fumes (open in a fume cupboard).</td></tr><tr><td>live/dead near IR</td><td>Life Technologies</td><td>L10119</td><td></td></tr><tr><td>vibratome</td><td>Technical Products International, Inc</td><td></td><td></td></tr><tr><td>coulture cup</td><td>Thermo-Fisher Scientific</td><td>SIE936</td><td>Mold for sectioning spheroids</td></tr><tr><td>hemocytometer</td><td>Sigma</td><td>Z359629</td><td></td></tr><tr><td>96-well tissue culture plate</td><td>Invitro</td><td>FAL353072</td><td></td></tr><tr><td>collagenase</td><td>Sigma</td><td>C5138&amp;nbsp;</td><td></td></tr><tr><td>confocal microscope</td><td>Leica</td><td>TCS SP5</td><td></td></tr><tr><td>Flow cytometer analyser</td><td>Becton Dickinson</td><td>LSRFortessa</td><td></td></tr><tr><td>volocity</td><td>PerkinElmer</td><td></td><td>Imaging software</td></tr><tr><td>flowjo</td><td>Tree Star</td><td></td><td>Flow cytometry software</td></tr><tr><td>Vaccuum grease</td><td>Sigma</td><td>Z273554</td><td></td></tr><tr><td>Mounting media</td><td>Vector Laboratories</td><td>H1000</td><td></td></tr><tr><td>FUCCI (commercial constructs)</td><td>Life Technologies</td><td>P36238</td><td>Transient transfection only</td></tr><tr><td>Cell strainer 70 &amp;#956;m</td><td>In Vitro</td><td>FAL352350</td><td></td></tr><tr><td>Round bottom 5 ml tubes (sterile)</td><td>In Vitro</td><td>FAL352003</td><td></td></tr><tr><td>Round bottom 5 ml tubes (non-sterile)</td><td>In Vitro</td><td>FAL352008</td><td></td></tr></tbody></table>

imaging- and flow cytometry-based analysis of cell position and the cell cycle in 3d melanoma spheroids

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions.

有产生肿瘤球状体的几种方法，该协议使用非贴壁生长的方法，其中细胞在琼脂或琼脂糖3,7,9中培养。 图1示出 C8161黑素瘤球体的后琼脂上3天的例子。 C8161球状体形成正常尺寸的球体的直径为500 - 600微米（平均值= 565，标准差= 19中，n = 3）后3天。其他黑素瘤细胞系，将形成球状体包括:WM793，WM983C，WM983B，WM164，1205LU（用该细胞系形成的球状体是不规则的，较不密集19）。 为了可视化三维球体模型内的各个细胞的细胞周期，C8161黑素瘤细胞转导的FUCCI系统7,15。由于球体的大尺寸（高达1毫米，直径在3天后在琼脂糖和24小时的生长在胶原为C8161），切片是在旋转椭圆体的中心可视化细胞的最佳选择。整个球状​​体（活的或固定的）可以通过共聚焦显微镜成像，但是共焦成像是仅能够穿透到大约150微米，因此球体的中间光学片只能在球状体的直径小于300微米的小而获得。 图2A，B和C示出了通过一个C8161 FUCCI球体的截面的一个例子。甲坏死核心，由G1期停滞细胞包围，在外层增殖细胞的梯度是明显的。识别和量化基于它们在球体和细胞周期状态位置细胞，进行半自动化图像分析。 图2D示出了FUCCI细胞口罩和球体轮廓， 而图2E示出了红色的数字的量化（G1）和绿色或黄色（S / G2 / M）细胞或者小于80微米的球体（外细胞）或大于80微米的球体边缘（内细胞）的边缘。这quantificati上显示，红细胞中的G1大大富集在旋转椭圆体的内部区域，如预期。 为了鉴定和潜在排序基于在球体它们的细胞周期的状态和位置的细胞，在与FUCCI系统组合的Hoechst染料扩散测定法使用 。整个球体用Hoechst染料结果孵育在染料的有限扩散进入旋转椭圆体的外层，这可用于给Hoechst阳性外层细胞从Hoechst通过流式细胞阴性内球体细胞中分离出来。 图2F示出了Hoechst的高达大约80微米的球体边缘渗透。通过肉眼的球体部分，并的染料浓度和温育时间优化分析获得的80微米从边缘的Hoechst的阳性距离，使得Hoechst的渗透密切标记增殖细胞中的外层（其主要发现少超过80微米的边缘），并没有渗透到的G1被捕area.The孵育时间用Hoechst可以改变，以获得更深或较浅的渗透。变Hoechst的渗透随时间在图3展示了一个例子。 图4A示出了门控为Hoechst的高和低种群的一个例子， 而图4C 和 D说明了选通为FUCCI红色，黄色和绿色的细胞。 图4B示出了量化为红（G1）的数量和绿色或黄色（S / G2 / M）细胞或者在Hoechst的高（外细胞）或Hoechst的低（内细胞）。再次这种技术表明，红细胞在G1变成（相似比照至图 2E所述的图像分析）大大富集在旋转椭圆体的内部区域。 <img alt="图1" src="/files/ftp_upload/53486/53486fig1.jpg" /> 数字1:C8161球体后琼脂培养3天代表相衬图像采取10倍的放大倍数C8161球体。比例尺等于100微米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53486/53486fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53486/53486fig2.jpg" /> 图2:C8161 FUCCI球体图像分析一个C8161 FUCCI的形象代表球体在20倍放大倍率拍摄vibratome部分。球体是三天琼脂糖，然后另外24小时在胶原基质中培养。共聚焦Z-片（A）Azami绿色，（B）Kusabira Orange2 和 （C）FUCCI覆盖。比例尺= 100微米。 （D）在Volocity创建红绿对象掩模，与在灰色球体轮廓。氩行表示从球体边缘80微米的距离。 （E）的红（G1）内的内的数字和绿色/黄色（S / G2 / M）细胞的定量（&gt; 80微米的球体边缘）和外（&lt;80微米的球体边缘）的区域。误差棒表示从来自2个独立实验4球体部分在SD。 （F）渗透的10μM赫斯特染色后1.5小时孵化。比例尺= 100微米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53486/53486fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53486/53486fig3.jpg" /> 图3:C8161球体的Hoechst染料扩散时间进程从全C8161的中间代表共焦的z片图像球状体指定的时间上琼脂糖培养4天，并孵育10μMHoechst的。在拍摄放大10倍。白色棒表示近似Hoechst的穿透深度。需要注意的是C8161琼脂糖球体比已在图2中被植入的胶原蛋白为24小时的C8161球体密集，从而减少染料渗透在同一时间点。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53486/53486fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53486/53486fig4.jpg" /> 图4:Hoechst公司的高和低的浇口20与球状体10μMHoechst的孵育后C8161 FUCCI球体流分析 （A）的实施例。蓝线表示未染色的控制，绿线表示完全染色赫斯特控制。内（Hoechst的低）和外（Hoechst的高）芘内（B）的定量的红色和绿色/黄色细胞的数目的opulations。误差棒表示从8个独立实验（包括活的排序和固定细胞分析）在SD。 FUCCI选通内（C）和外部（D）球体群体的例子。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53486/53486fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids at JoVE.com

Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

We describe two complementary methods using the fluorescence ubiquitination cell cycle indicator (FUCCI) and image analysis or flow cytometry to identify and isolate cells in the inner G1 arrested and outer proliferating regions of 3D spheroids.

单元位置的Imaging-和流式细胞仪为基础的分析和3D球体黑色素瘤细胞周期

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to ...

imaging-flow-cytometry-based-analysis-cell-position-cell-cycle-3d

Universidad San Sebastian

Research

JoVE Journal

Medicine

12.9K 次观看.  The Centenary Institute. We describe two complementary methods using the fluorescence ubiquitination cell cycle indicator (FUCCI) and image analysis or flow cytometry to identify and isolate cells in the inner G1 arrested and outer proliferating regions of 3D spheroids. 

视频: Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

A 3D Organotypic Melanoma Spheroid Skin Model

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased metastatic potential. Recently, mono-chemotherapies continue to improve by melanoma specific combination therapies with targeted kinase inhibitors. Still, metastatic melanoma remains a life-threatening disease because tumors exhibit primary resistance or develop resistance to novel therapies, thereby regaining tumorigenic capacity. In order to improve the therapeutic success of malignant melanoma, the determination of molecular mechanisms conferring resistance against conventional treatment approaches is necessary; however, it requires innovative cellular in vitro models. Here, we introduce an in vitro three-dimensional (3D) organotypic melanoma spheroid model that can portray the in vivo architecture of malignant melanoma and may warrant new insights into intra-tumoral as well as tumor-host interactions. The model incorporates defined numbers of mature and differentiated melanoma spheroids in a 3D human full skin reconstruction model consisting of primary skin cells. The cellular composition and differentiation status of the embedded melanoma spheroids is similar to the one of cutaneous melanoma metastasis in vivo. Using this organotypic melanoma spheroid model as a drug screening platform may support the identification of responders to selected combination therapies, while sparing the unnecessary treatment burden for non-responders, thereby increasing the benefit of therapeutic interventions.

Here, we present a protocol to generate a 3D organotypic melanoma spheroid skin model that recapitulates both the architecture and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention.

3D 脊髓黑色素瘤球形皮肤模型

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased ...

科研

癌症研究

Longitudinal Morphological and Physiological Monitoring of Three-dimensional Tumor Spheroids Using Optical Coherence Tomography

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, high-throughput imaging modalities utilizing bright field or fluorescence detection, are unable to resolve the overall 3D structure of the tumor spheroid due to limited light penetration, diffusion of fluorescent dyes and depth-resolvability. Recently, our lab demonstrated the use of optical coherence tomography (OCT), a label-free and non-destructive 3D imaging modality, to perform longitudinal characterization of multicellular tumor spheroids in a 96-well plate. OCT was capable of obtaining 3D morphological and physiological information of tumor spheroids growing up to about 600 &#181;m in height. In this article, we demonstrate a high-throughput OCT (HT-OCT) imaging system that scans the whole multi-well plate and obtains 3D OCT data of tumor spheroids automatically. We describe the details of the HT-OCT system and construction guidelines in the protocol. From the 3D OCT data, one can visualize the overall structure of the spheroid with 3D rendered and orthogonal slices, characterize the longitudinal growth curve of the tumor spheroid based on the morphological information of size and volume, and monitor the growth of the dead-cell regions in the tumor spheroid based on optical intrinsic attenuation contrast. We show that HT-OCT can be used as a high-throughput imaging modality for drug screening as well as characterizing biofabricated samples.

Optical coherence tomography (OCT), a three-dimensional imaging technology, was used to monitor and characterize the growth kinetics of multicellular tumor spheroids. Precise volumetric quantification of tumor spheroids using a voxel counting approach, and label-free dead tissue detection in the spheroids based on intrinsic optical attenuation contrast, were demonstrated.

三维肿瘤球体的纵向形态和生理监测

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, ...

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of anti-cancer therapeutics in a physiologically representative microenvironment. We outline the formation of patient derived cancer stem cell spheroids, as well as, the manipulation of these spheroids for thorough analysis following drug treatment. Specifically, we describe collection of spheroid morphology, proliferation, viability, drug toxicity, cell phenotype and cell localization data. This protocol focuses heavily on analysis techniques that are easily implemented using the 384-well hanging drop platform, making it ideal for high throughput drug screening. While we emphasize the importance of this model in ovarian cancer studies and cancer stem cell research, the 384-well platform is amenable to research of other cancer types and disease models, extending the utility of the platform to many fields. By improving the speed of personalized drug screening and the quality of screening results through easily implemented physiologically representative 3D cultures, this platform is predicted to aid in the development of new therapeutics and patient-specific treatment strategies, and thus have wide-reaching clinical impact.

This protocol describes generation of patient-derived spheroids, and downstream analysis including quantification of proliferation, cytotoxicity testing, flow cytometry, immunofluorescence staining and confocal imaging, in order to assess drug candidates&#8217; potential as anti-neoplastic therapeutics. This protocol supports precision medicine with identification of specific drugs for each patient and stage of disease.

生理患者衍生的3D类血球体用于抗肿瘤药物筛选,以靶向癌症干细胞

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of ...

Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. The power of this preparation lies in its ability to mimic many aspects of the in vivo conditions of tumors while being fast, cheap, and versatile enough to allow relatively high-throughput screening. The spheroid culture conditions can recapitulate the physico-chemical gradients in a tumor, including the increasing extracellular acidity, increased lactate, and decreasing glucose and oxygen availability, from the spheroid periphery to its core. Also, the mechanical properties and cell-cell interactions of in vivo tumors are in part mimicked by this model. The specific properties and consequently the optimal growth conditions, of 3D spheroids, differ widely between different types of cancer cells. Furthermore, the assessment of cell viability and death in 3D spheroids requires methods that differ in part from those employed for 2D cultures. Here we describe several protocols for preparing 3D spheroids of cancer cells, and for using such cultures to assess cell viability and death in the context of evaluating the efficacy of anticancer drugs.

Here, we present several simple methods for evaluating viability and death in 3D cancer cell spheroids, which mimic the physico-chemical gradients of in vivo tumors much better than the 2D culture. The spheroid model, therefore, allows evaluation of the cancer drug efficacy with improved translation to in vivo conditions.

评估癌细胞3D球形培养中的细胞活力和死亡

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. ...

A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor stroma, host fibroblasts are stimulated by cancer cells to differentiate. Thus, they acquire a phenotype that contributes to the tumor microenvironment and supports tumor progression. By using the spheroid model, we have set up such a 3D in vitro model system, in which we analyzed the role of laminin-332 and its receptor integrin &#945;3&#946;1 in this differentiation process. This spheroid model system not only reproduces the tumor microenvironment conditions in a more accurate way, but also is a very versatile model since it allows different downstream studies, such as immunofluorescent staining of both intra- and extracellular markers, as well as deposited extracellular matrix proteins. Moreover, transcriptional analyses by qPCR, flow cytometry and cellular invasion can be studied with this model. Here, we describe a protocol of a spheroid model to assess the role of CAFs&#39; integrin &#945;3&#946;1 and its ectopically deposited ligand, laminin-332, in differentiation and in supporting the invasion of pancreatic cancer cells.

The goal of this protocol is to establish a 3D in vitro model to study the differentiation of cancer-associated fibroblasts (CAFs) in a tumor bulk-like environment, which can be addressed in different analysis systems, such as immunofluorescence, transcriptional analysis and life cell imaging.

3D球形模型作为癌症相关纤维细胞分化和入侵体内研究的更多生理系统

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor ...

A Novel Stromal Fibroblast-Modulated 3D Tumor Spheroid Model for Studying Tumor-Stroma Interaction and Drug Discovery

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro model in the study of cancer stem/initiating cells, preclinical cancer research, and drug screening. The 3D spheroid models are superior to conventional tumor cell culture and reproduce some important characters of real solid tumors. However, conventional 3D tumor spheroids are made up exclusively of tumor cells. They lack the participation of tumor stromal cells and have insufficient extracellular matrix (ECM) deposition, thus only partially mimicking the in vivo conditions of tumor tissues. We established a new multicellular 3D spheroid model composed of tumor cells and stromal fibroblasts that better mimics the in vivo heterogeneous tumor microenvironment and its native desmoplasia. The formation of spheroids is strictly regulated by the tumor stromal fibroblasts and is determined by the activity of certain crucial intracellular signaling pathways (e.g., Notch signaling) in stromal fibroblasts. In this article, we present the techniques for coculture of tumor cells-stromal fibroblasts, time-lapse imaging to visualize cell-cell interactions, and confocal microscopy to display the 3D architectural features of the spheroids. We also show two examples of the practical application of this 3D spheroid model. This novel multicellular 3D spheroid model offers a useful platform for studying tumor-stroma interaction, elucidating how stromal fibroblasts regulate cancer stem/initiating cells, which determine tumor progression and aggressiveness, and exploring involvement of stromal reaction in cancer drug sensitivity and resistance. This platform can also be a pertinent in vitro model for drug discovery.

A novel three-dimensional spheroid model based on the heterotypic interaction of tumor cells and stromal fibroblasts is established. Here, we present coculture of tumor cells and stromal fibroblasts, time-lapse imaging, and confocal microscopy to visualize the formation of spheroids. This three-dimensional model offers a pertinent platform to study tumor-stroma interactions and test cancer therapeutics.

用于研究肿瘤-斯特罗马相互作用和药物发现的新型斯特罗姆纤维细胞-模组3D肿瘤球体模型

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro ...

Rapid Optical Clearing for Semi-High-Throughput Analysis of Tumor Spheroids

Tumor spheroids are fast becoming commonplace in basic cancer research and drug development. Obtaining data regarding protein expression within the spheroid at the cellular level is important for analysis, yet existing techniques are often expensive, laborious, use non-standard equipment, cause significant size distortion, or are limited to relatively small spheroids. This protocol presents a new method of mounting and clearing spheroids that address these issues while allowing for confocal analysis of the inner structure of spheroids. In contrast to existing approaches, this protocol provides for rapid mounting and clearing of a large number of spheroids using standard equipment and laboratory supplies. Mounting spheroids in a pH-neutral agarose-PBS gel solution before introducing a refractive-index-matched clearing solution minimizes size distortion common to other similar techniques. This allows for detailed quantitative and statistical analysis where the accuracy of size measurements is paramount. Furthermore, compared to liquid clearing solutions, the agarose gel technique keeps spheroids fixed in place, allowing for the collection of three-dimensional (3D) confocal images. The present article elaborates how the method yields high-quality two- and 3D images that provide information about inter-cell variability and inner spheroid structure.

Tumor spheroids are becoming increasingly utilized to assess tumor cell-microenvironment interactions and therapy response. The present protocol describes a robust but simple method for semi-high-throughput imaging of 3D tumor spheroids using rapid optical clearing.

用于肿瘤球体半高通量分析的快速光学清除

Tumor spheroids are fast becoming commonplace in basic cancer research and drug development. Obtaining data regarding protein expression within the ...

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 ...

医学

<meta charset="utf8"></head><body>检查 3D 胶原基质设置中的巨噬细胞-肿瘤球状体相互作用

A Macrophage-Tumor Spheroid Co-Invasion Assay

The interaction of immune and cancer cells and their respective impact on metastasis represents an important aspect of cancer research. So far, only a few protocols are available that allow an in vitro approximation of the in vivo situation. Here, we present a novel approach to observing the impact of human macrophages on the invasiveness of cancer cells, using tumor spheroids of H1299 non-small cell lung carcinoma cells embedded in a three-dimensional (3D) collagen I matrix. With this co-cultivation setup, we tested the impact of small interfering RNA (siRNA)-based depletion of regulatory factors in macrophages on the 3D invasion of cancer cells from the tumor spheroid compared to controls. This method allows the determination of different parameters, such as spheroid area or the number of invading cancer cells, and thus, to detect differences in cancer cell invasion. In this article, we present the respective setup, discuss the subsequent analysis, as well as the advantages and potential pitfalls of this method.

<meta charset="utf8"></head><body>巨噬细胞-肿瘤球状体共侵袭测定

Here, we present a protocol to investigate the interaction of primary human monocyte-derived macrophages with a tumor spheroid in a three-dimensional (3D) collagen I matrix, with the possibility to compare the impact of soluble and physical properties of the microenvironment on cell invasion.

巨噬细胞-肿瘤球状体共侵袭测定

The interaction of immune and cancer cells and their respective impact on metastasis represents an important aspect of cancer research. So far, only a few ...

单元位置的Imaging-和流式细胞仪为基础的分析和3D球体黑色素瘤细胞周期

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用于肿瘤球体半高通量分析的快速光学清除

三维（3D）肿瘤球体侵袭试验

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巨噬细胞-肿瘤球状体共侵袭测定