This work was funded by a Natural Sciences and Engineering Research Council (NSERC) Canada grant to MGM. JR is a NSERC-Vanier student fellow.

注意:下面的每个步骤应在无菌条件下在层流罩的要求用于哺乳动物细胞培养物中进行的。此外，为了获得最佳无菌细胞培养的一般准则适用， 例如 ，丢弃提示的任何时间，他们可能会导致交叉污染，减少了时间单元的数量进行时，整个媒体的变化，妥善而是轻轻搅拌所有人都暴露在空气中细胞悬浮液，以确保其均匀移液等。此外，插入件是一种塑料制品的需要特殊处理。首先，当插入被操纵，避免接触脆弱的膜，这泪水易被，因此可能会危及实验。此外，它是不适合于执行所述细胞培养基的真空抽吸，因为有穿孔的膜或离解的贴壁细胞的危险。接着，插入件中的多孔组织培养板松散挂的，因此，必须谨慎时莫采用咏的塑料制品或移液时，为了避免解离贴壁细胞。此外，使用具有大孔径大小的插入时，存在的细胞培养基渗透通过该膜，因此，频繁地监测液位是很重要的一个可能性。最后，请注意以下方案被设计用于贴壁细胞，并且需要较小的修改，以适合于悬浮细胞。 1.在进行插入共培养实验的一般准则<ol><li>在插入种子细胞类型＃1 <ol><li>解开从包装插入件。 </li><li>放置插入件以适当的尺寸的空多孔的组织培养板。这样做，把手用镊子刀片的最上边缘。 </li><li>为了提高附着和粘附细胞的扩散，向播种之前调节与细胞培养基中插入。为此，覆盖膜的细胞培养mediu整个表面中号使用微量。 注:按要求执行此操作尽可能多的插入。 </li><li>上盘更换盖子，在相同条件下孵育至少1小时或O / N（通常在37℃，5％-10％ 的 CO 2）。 </li><li>当刀片调节，除去所有使用微量的细胞培养基。丢弃使用过网上平台。 </li><li>作为在多孔板中相同的方式新鲜细胞培养基的种子细胞类型＃1。这样做，绘制用微量的细胞悬浮液的适当体积和分配在插入液体。 注:根据需要准备尽可能多的插入。 </li><li>播种所有插入后，轻轻摇动左右的板，然后来回，以均匀分布的细胞。避免使打圈，因为这会导致细胞中的插入物的中心积聚。 </li><li>放置在平板上的盖以及由按蜂窝要求（通常为37℃，5-10％的CO指定孵育<suB&gt; 2）。 </li></ol></li><li>种子细胞类型中排名第2多孔组织培养板<ol><li>根据第1节中的新鲜细胞培养基的种子细胞类型＃2。 </li><li>根据需要对刀片的数目准备许多井。摇动平板在步骤1.1.7），以确保将细胞均匀地分布在孔中。 </li><li>放置在板盖和在相同条件下孵育（通常在37℃，5％-10％ 的 CO 2）。 </li></ol></li><li>在插入清爽中<ol><li>用微量，除去部分或全部的细胞培养基，在含细胞类型＃1的插入。丢弃使用过网上平台。 </li><li>绘制新鲜细胞培养基的适当体积。轻轻靠在插入件的内壁的前端，慢慢地分配所述细胞培养基。 </li><li>放置在板盖和孵化按照步骤1.1.4。 </li></ol></li><li>在多孔组织培养皿中令人耳目一新<ol><li>使用micropi佩特，除去部分或全部的含细胞类型＃2的孔中的细胞培养基。丢弃使用过网上平台。 </li><li>绘制新鲜细胞培养基的适当体积。搁置在井的内壁的前端，慢慢地分配所述细胞培养基。 </li><li>放置在平板上的盖子，并根据先前建立的细胞培养物中的协议孵育。 </li></ol></li><li>将含有细胞类型＃1至含细胞类型＃2多孔组织培养板中的插入。 注意:当两种类型的细胞已经达到合适的成长阶段执行此步骤。 <ol><li>之前的插入物转移入井中，进行必要的媒体的变化，如先前在步骤1.3）和1.4中所述）。 注意:在这一点上，它由插入件制造商的说明指定以分配媒体的适当体积中两个隔室是重要的。 </li><li>使用镊子，把手含细胞t时刀片的最上边缘YPE＃1，轻轻将其放置在适当的孔含有细胞类型＃2。 </li><li>转移所有插入后，检查气泡的存在插入的膜下。 注:气泡防止横跨所述嵌件的膜的任何交换，并且可以危及整个实验。 </li><li>如果存在气泡，轻轻地从井用镊子提起插入物和沉浸放回细胞培养基。气泡会消失。他们是否仍然存在，尝试轻轻一角度浸渍插入放回细胞培养基。 注意:不要敲打或搅拌刀片，以避免解离贴壁细胞。 </li><li>除去所有的气泡和检查的介质在这两个隔室的体积，放置在平板上的盖子和孵化后。 </li></ol></li><li>在共培养体系清爽媒体 注:虽然膜容易使刀片之间的媒体交流和幸福，刷新介质中进行由于通过扩散独自以达到在上部和下部隔室的平衡所需要的时间两个室可以是相当长的。 <ol><li>在含有细胞类型＃1的插入清凉介质以相同的方式完成，如步骤1.3）。 </li><li>刷新在含细胞类型＃2孔介质，插入物轻轻推向一侧创造足够宽，以容纳一个枪头的空间。刷新介质如在步骤1.4）。 </li><li>检查气泡的存在和通过1.5.5验证卷按步骤1.5.3））。 </li></ol></li></ol> 2.实施例:测量由对神经元PC12细胞LPS活化的N9小胶质细胞分泌的细胞因子的影响注:以下步骤是专为特定瓶，好，菜的大小。然而，该协议可以定制任何塑料制品尺寸。对于媒体和组成见材料表。 <ol><li>播种和多分化PC12细胞孔组织培养板<ol><li>暖常规PC12细胞培养基，PC12分化培养基和胰蛋白酶-EDTA在37℃水浴中。 </li><li>使用PC12细胞在60-80％汇合从一个75cm 2的烧瓶中。 </li><li>带有15mm巴斯德吸管，执行烧瓶整个细胞培养液中的负压吸引。 </li><li>轻轻漂洗单层细胞用5毫升的无菌磷酸盐缓冲盐水中，用巴斯德吸管除去液体。要小心，不要在此步骤中分离细胞。 </li><li>覆盖用3ml胰蛋白酶-EDTA的细胞单层，并在37°C孵育2-3分钟。 </li><li>确保所有的细胞在显微镜下分离。如果很少细胞浮动，孵育时间最长为5分钟的较​​长的时间。 </li><li>加入10 mL常规的PC12细胞培养基以灭活胰蛋白酶-EDTA。 </li><li>用10毫升吸管，同时确保大多数细胞从底部离解Ò轻轻磨碎F中的烧瓶中。避免在细胞悬浮液中产生气泡。 </li><li>用同样的10毫升吸管，在50ml离心管中转移细胞悬浮液。 </li><li>离心1分钟3,200 XG </li><li>用巴氏吸管弃去上清液同时小心不要打扰颗粒。 </li><li>加入10 mL常规PC12细胞培养基用10毫升吸管。 注:如果颗粒异常大或小本音量可调节。 </li><li>用同样的10毫升吸管磨碎以均质沉淀。 PC12细胞常聚集在一起，所以至少20吹打向上和downare必要的。 注意:当剧烈研制过程是必要的，避免在细胞悬浮液中产生气泡。 </li><li>在1.5ml管中，准备在台盼蓝的细胞悬浮液的适当稀释。算使用根据先前建立的协议74血球细胞。 </li><li>在一个单独的50ml管中，分裂细胞悬浮液与PC12分化培养基以得到稀释的细胞悬浮液适合于从24孔板接种孔（30,000¢/厘米2，0.6毫升，每根据制造商的协议以及）。 注:24孔板，必须预先涂有胶原蛋白通过预先建立的方案75指定。 </li><li>每孔使用微量分发0.6毫升细胞悬浮液。 </li><li>当所有的井去籽，摇动平板在步骤1.1.7）。 </li><li>以允许PC12细胞的正常分化，在37℃下在对PC12分化培养基15,16在5％CO 2湿润气氛中进行共培养实验之前孵育24孔板为7-9天。 </li><li>通过除去液体的一半，并用等体积的新鲜的PC12分化培养基替换它执行介质改变每隔一天。 </li></ol></li><li>在插入播种N9小胶质细胞和用LPS治疗<ol><li>在进行共培养实验的前一天，预先处理的PTFE0.4μm的孔隙刀片与常规的N9细胞培养基，以优化细胞粘附，以下步骤1.1.1）通过1.1.4） </li><li>暖常规N9细胞培养基和胰蛋白酶-EDTA在37℃水浴中。 </li><li>与此同时，重约10毫克的LPS在1.5ml管供以后使用。 注意:由于LPS是一种强效的促炎内毒素，并且需要特别的安全防范措施，眼镜，手套和粒状呼吸器强烈建议。 </li><li>使用N9细胞在80-90％汇合时从一个75cm 2的烧瓶中。 </li><li>按照步骤2.1.3）至2.1.14）。始终使用常规N9细胞培养液中，而不是常规的PC12细胞培养液中。还注意到，N9小胶质细胞不聚集在一起多达PC12细胞做的，所以较少的移液上下可以是在步骤2.1.13必要）。 </li><li>在一个单独的50ml管中，分割用N9细胞培养基邻的细胞悬浮液btain适当播种设计24孔板容纳刀片（60,000¢/厘米2，0.05根据制造商的协议毫升每插入，用于膜表面积见生产商信息）的稀释细胞悬液。 注意:这些插入物已涂覆有由生产胶原蛋白和细胞粘附进行了优化。 </li><li>分发0.05毫升使用微量每个刀片的细胞悬浮液。 </li><li>当所有的刀片是种子，岩盘如步骤1.1.7）。 </li><li>执行使用N9处理介质，以获得4微克/毫升，2微克/毫升和1微克/毫升工作溶液的LPS的连续稀释液。 </li><li>吸管0.05毫升4微克/毫升的工作在一集合插入以得到的2微克/毫升的最终的稀释溶液。 </li><li>重复步骤2.2.10），用于在不同组的插入另外两个工作溶液，得到1微克的最终稀释度/ ml和0.5微克/分别毫升</li><li>在cubate含24小时的插入所述板在37℃在一个5％CO 2的潮湿气氛允许N9小胶质细胞活化用LPS进行共培养实验之前。 </li></ol></li><li>联合培养的神经元PC12细胞与小胶质N9 <ol><li>在分化的PC12细胞的7-9天，激活后在37℃水浴中通过温暖的N9处理介质和PC12处理介质的LPS孵育的24小时的N9小胶质细胞。 </li><li>执行为N9小胶质细胞共介质变化如在步骤1.3），通过0.1毫升N9处理介质的更换整个用过的介质。为此，每insert.This必须除去内毒素的所有痕迹，只留下激活N9小胶质细胞中的插入件。 </li><li>执行对神经元PC12细胞如​​在步骤1.4，总介质变化）。在一个5％CO 2的潮湿气氛转移镶入24孔板如在步骤1.5）.Incubate的N9-PC12共培养24小时或48小时，在37℃。 </li><li> 24后小时或48小时，收获上清液和/或细胞毒性，酶联免疫测定法中的细胞（ELISA），Western印迹，或其它测定。 </li></ol></li></ol>

The authors have nothing to disclose.

任何插入共培养体系实验的最关键的一步其实住在选择合适的插入使用。孔径和膜材料必须考虑到彻底帐户，没有忘记考虑将被接种的细胞的类型和实验的目的。例如，趋化性测定法可使用相同类型的膜的比细胞共培养以分析由在不存在细胞 - 细胞接触的分泌的可溶性因子诱导的细胞行为调制。然而，这两种类型的实验需要不同的孔径大小:较大的为前，允许细胞迁移，以及较小的为后者，以排除...

组织，器官或系统的体外研究是为了简化构成多细胞生物的几个细胞亚型之间存在的非常复杂的关系的一种尝试。实际上， 在体外研究使得能够获得单细胞群的一个详细的了解。有体外实验进行两个主要的优点:1）降低细胞相互作用，以及2）容易地操纵细胞环境的能力。因此，这两个优点已使科学家能够预测在体内的特定细胞类型的行为，导致以调节在整个生物体外在影响结果的能力。在这个意义上， 体外细胞培养往往可以作为连接基础和应用生命科学的桥梁。尽管如此，也有在体外工作的若干缺点，其中最重要的一种被某一个预留可以住在生理人观察到的表型的相关性。事实上，当一个单一细胞类型生长容器中，该培养失去，在不同程度，与其他类型的细胞，其以从组织和原籍有机体，和内锚体液环境贡献其细胞 - 细胞连接这使得它的组织坚持一个特定的三维结构有时细胞功能的关键。 细胞间关系的问题已被混合培养技术的发展解决。在该方法中，两个或更多个细胞群体在同一培养容器生长在一起。然而，这些混合文化具有重要的不便。一方面，一些细胞亚型物理上不与起源组织彼此互动，仅仅依靠通过分泌可溶性因子及受体附近持续旁分泌通信。这对于依赖于近端细胞因子信号若干炎症过程的情况。在混合Cultures，物理相互作用是不可避免的，使得不可能研究在不存在的细胞 - 细胞接触，可以产生改变的结果的旁分泌的通信。另一方面，从混合群体内实现细胞特异性的解释变得不使用苛刻的分离技术，可以显著影响的结果是不可行的。 为了解决这些重要问题，使用条件培养基的已发展为允许条块文化和旁分泌信号传导的研究的技术。这种方法需要一种细胞类型，因此命名条件培养基的上清液转移到含有细胞的另一群井。然而，一个重要的缺点是短命的分子不存活足够长的条件培养基被转移到细胞的第二群体的孔中。即使是长寿命的分子会大大摊薄随着时间的推移，由于扩散。此外，这两种细胞居民只能参加单向旁分泌通信，而不是主动的双向通信。这导致了不存在，因为它们在体内存在即在重新准确多关系的重要的反馈信令。 作为结果和由需要更好地模拟在 体外细胞环境体内条件的原始驱动，在细胞培养技术若干进步已多年来实现。其中最显著进步已利用与微孔膜为compartmentalizing细胞培养物，用于第一时间由Grobstein可渗透支撑于1953年1，这种可渗透的载体已多年来定制，以适应众多细胞类型，也可以使用在几个不同的应用程序。如今，这些支撑存在，旨在从多孔组织培养板或断路器操作过井休息空心插入LAR菜肴。在共培养系统中，插入包含一种细胞类型，而孔或培养皿包含其他细胞群体，从而来研究它们的体液环境（ 图1）的细胞的两个不同群体的贡献。其结果是，细胞极性（基底VS心尖分泌或信号接收）被保留，从而赋予插入共培养系统在混合培养物和条件培养基中的技术的一个重要优点。几种类型的膜材料都是可用的，最常见的是聚酯（PET），聚碳酸酯（PC）或胶原包被的聚四氟乙烯（PTFE），以及它们在不同的孔尺寸为0.4微米至12.0微米的存在。这些品种的材料和孔径提供刀片施加相关的光学特性，膜厚度和细胞粘附，使它们在不同的水平以下实用可变特征的频谱用途不限于: -studying细胞分化，胚胎发育，肿瘤的转移和通过渗透膜趋化性测定法创伤修复; 通过上皮或内皮单层评估其运输-evaluating药物渗透上可渗透支撑培养，; -performing细胞共培养以分析由在不存在细胞 - 细胞接触的分泌的可溶性因子诱导的细胞行为调制。 本文的目的是描述一般方法准则以满足上述第三功能，即评估通过使用插入共培养体系中没有细胞 - 细胞接触的分泌的可溶性因子介导的细胞的变化。的研究几种不同的领域作出为了回答相关的分泌的可溶性因子对细胞群的影响的问题使用刀片共培养系统。的确，在各级调制细胞行为旁分泌信号是在所有的组织中的相关和系统，这使得插入物共培养系统不可或缺，以确保在这些领域的进步。相反，利用插入物可以确认的信号转导是由直接细胞 - 细胞接触，而不是由分泌的因子。一插入物的最重要的用途是在炎症研究2-14，其中分泌的细胞因子的作用在免疫的各种细胞的玩家进行评价。特别是，炎症在中枢神经系统（CNS）的研究已经大大从插入共培养研究中，这些都使更好地限定在驱动神经炎症15-21神经元和小胶质细胞的不同旁分泌作用中获益。这些系统也被设计来研究依赖于它们，以减少或抑制促炎因子22-26的分泌能力的分子的抗炎潜力。关于癌症27-31，特别机制底层的血管生成32-34和inflammati研究在肿瘤35-42，也插入共培养体系中受益。此外，可溶性因素的推动分化和一些研究使用刀片来回答在特定领域的问题43-50过程最重要的。在中枢神经系统，看到神经组织具有非常有限的更新能力，神经营养的研究和神经保护是根本，目前已广泛用在共同培养系统51-56利用干细胞的保证。此外，刀片也被用来作为不同领域的肾脏57,58，内皮细胞的相互作用和血管生成59-62，细胞凋亡信号63-65，炎症肥胖和代谢综合征22,23,66-67，内耳毛细胞有保护作用68,69，甚至致病真菌和70,71 72,73寄生虫。 本文以建立一个experim提供了常用方法指南耳鼻喉科鉴于评估通过插入共培养体系分泌的可溶性因子介导的细胞的变化。特别是，我们将我们的注意力集中于神经细胞共培养他们学神经炎过程中的用途。鉴于实验十分广阔频谱接入成为可能试点，令人难以忍受来弥补这一细胞培养技术的各个方面。作为一个例子，特定的协议以测量由脂多糖（LPS）分泌的细胞因子的作用活化的对神经元PC12细胞N9小胶质细胞将被详细描述的，提供插入物共培养方法的一个具体的了解。

<table><tbody><tr><td>RPMI-1640 medium</td><td>Sigma</td><td>R8755</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium/Nutrient Mixture F-12 Ham</td><td>Sigma</td><td>D6421</td><td>Warm in 37 &amp;deg;C water bath before use, must be supplemented with 0.365 gm/L L-glutamine</td></tr><tr><td>Horse serum</td><td>ATCC</td><td>30-2040</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Fetal bovine serum</td><td>MultiCell</td><td>80350</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Nerve Growth Factor-7S from murine submaxillary gland</td><td>Sigma</td><td>N0513</td><td>Reconstitute the lyophilized powder in a solution of buffered saline or tissue culture medium containing 0.1&amp;ndash;1.0% bovine serum albumin or 1-10% serum</td></tr><tr><td>Trypsin-EDTA solution</td><td>Sigma</td><td>T3924</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Lipopolysaccharides from&amp;nbsp;Escherichia coli&amp;nbsp;055:B5</td><td>Sigma</td><td>L2880</td><td>Toxic</td></tr><tr><td>Cell culture inserts for use with 24-well plates</td><td>BD Falcon</td><td>353095</td><td>0.4 &amp;mu;m pores</td></tr><tr><td>24-well plates</td><td>TrueLine</td><td>TR5002</td><td>Coat with collagen before use</td></tr><tr><td>Routine PC12 cell culture medium</td><td></td><td></td><td>Routine N9 cell culture medium</td></tr><tr><td>-&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 85% RPMI medium</td><td></td><td></td><td>-&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 90% DMEM-F12 medium</td></tr><tr><td>-&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 10% heat-inactivated horse serum</td><td></td><td></td><td>-&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 10% heat-inactivated horse serum</td></tr><tr><td>-&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 5% heat-inactivated fetal bovine serum</td><td></td><td></td><td></td></tr><tr><td>PC12 differentiation medium</td><td></td><td></td><td>N9 treatment medium</td></tr><tr><td>-&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 99% RPMI medium</td><td></td><td></td><td>-&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 99% DMEM-F12 medium</td></tr><tr><td>-&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 1% heat-inactivated fetal bovine serum</td><td></td><td></td><td>-&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 1% heat-inactivated fetal bovine serum</td></tr><tr><td>-&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 50 ng/mL nerve growth factor</td><td></td><td></td><td></td></tr><tr><td>PC12 treatment medium</td><td></td><td></td><td></td></tr><tr><td>-&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 99% RPMI medium</td><td></td><td></td><td></td></tr><tr><td>-&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 1% heat-inactivated fetal bovine serum</td><td></td><td></td><td></td></tr></tbody></table>

development of an insert co-culture system of two cellular types in the absence of cell-cell contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

使用插入共培养系统，是在展示中枢神经系统的细胞不同玩家之间的关系，旁分泌神经炎症过程的研究尤为相关。免疫力在CNS被称为小胶质固有细胞监视它们在其静止支链状态（ 图2A）的环境，并能够感测扰动主要完成可能麻烦必要进行适当的神经元功能76-78的非常宝贵的动态平衡。小胶质细胞活化，其特征在于采用一个变形虫形状（ 图2B）</str...

Watch this Scientific Journal Video about Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact at JoVE.com

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

在细胞间接触缺失的两个蜂窝型的插入共培养系统的开发

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...

development-an-insert-co-culture-system-two-cellular-types-absence

Research

JoVE Journal

Biology

27.8K 次观看.  University of Québec. In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

视频: Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events leading to stromal activation and establishment of the tumor microenvironment (TME). Several in vitro and in vivo models of the TME have been developed; however, in general these models do not readily permit isolation of individual cell populations, under non-perturbing conditions, for further study. To circumvent this difficulty, we have employed an in vitro TME model using a cell growth substrate consisting of a permeable microporous membrane insert that permits simple generation of highly enriched cell populations grown intimately, yet separately, on either side of the insert&#39;s membrane for extended co-culture times. Through use of this model, we are capable of generating greatly enriched cancer-associated fibroblast (CAF) populations from normal diploid human fibroblasts following co-culture (120 hr) with highly metastatic human breast carcinoma cells, without the use of fluorescent tagging and/or cell sorting. Additionally, by modulating the pore-size of the insert, we can control for the mode of intercellular communication (e.g., gap-junction communication, secreted factors) between the two heterotypic cell populations, which permits investigation of the mechanisms underlying the development of the TME, including the role of gap-junction permeability. This model serves as a valuable tool in enhancing our understanding of the initial events leading to cancer-stroma initiation, the early evolution of the TME, and the modulating effect of the stroma on the responses of cancer cells to therapeutic agents.

We adapted a permeable microporous membrane insert to mimic the tumor microenvironment (TME). The model consists of a mixed cell culture, allows simplified generation of highly enriched individual cell populations without using fluorescent tagging or cell sorting, and permits studying intercellular communication within the TME under normal or stress conditions.

肿瘤微环境的模仿:产生富集的细胞群及侦查间通讯的简单方法

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events ...

科研

癌症研究

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

利用双水相系统的细胞共培养图案

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

生物工程

Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies of endothelial-epithelial bilayers have traditionally relied on permeable support models that enable bilayer culture, but permeable supports are limited in their ability to replicate the diversity of human basement membranes. In contrast, hydrogel models that require chemical synthesis are highly tunable and allow for modifications of both the material stiffness and the biochemical composition via incorporation of biomimetic peptides or proteins. However, traditional hydrogel models are limited in functionality because they lack pores for cell-cell contacts and functional in vitro migration studies. Additionally, due to the thickness of traditional hydrogels, incorporation of pores that span the entire thickness of hydrogels has been challenging. In the present study, we use poly-(ethylene-glycol) (PEG) hydrogels and a novel zinc oxide templating method to address the previous shortcomings of biomimetic hydrogels. As a result, we present an ultrathin, basement membrane-like hydrogel that permits the culture of confluent cellular bilayers on a customizable scaffold with variable pore architectures, mechanical properties, and biochemical composition.

Current bilayer culture models do not allow for functional in vitro studies that mimic in vivo microenvironments. Using polyethylene glycol and a zinc oxide templating method, this protocol describes the development of an ultrathin biomimetic basement membrane with tunable stiffness, porosity, and biochemical composition that closely mimics in vivo extracellular matrices.

超薄 Porated 弹性水凝胶作为双细胞培养的仿生基底膜

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies ...

A Co-culture Method to Investigate the Crosstalk Between X-ray Irradiated Caco-2 Cells and PBMC

The protocol adopted in this work aims at unraveling how X-rays perturb the functioning of the intestinal barrier, focusing on the interplay between colorectal tumor cells and the immune system. Colorectal carcinoma is among the most common type of cancer, typically treated by surgery, chemotherapy, and radiotherapy. Advantages of radiotherapy in targeting the tumor are well known. However, even limited exposures of healthy tissues are of great concern, particularly regarding the effects on the intestinal barrier and the immune system. The adopted setup allows to study the interplay between two cell populations in a condition more similar to the physiological one, when compared to normal cell cultures. For this purpose, we resort to different techniques and we used an in vitro co-culture model, based on Caco-2 cells differentiated as a monolayer and PBMC, sharing the same culture medium. This protocol has been developed to focus on both macroscopic effects, i.e. cell viability and Trans-Epithelial Electrical Resistance (TEER), and, through western blot, molecular alterations, i.e. the activation of inflammatory pathway in immune cells and the tight junction protein expression in Caco-2 cells. Initial evaluation of radiation effects on Caco-2 cell viability was assessed via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Trypan blue assays, while TEER was measured at fixed time intervals through an ohmmeter specifically designed for co-culture systems. In this way, the effects due to radiation, the presence of Peripheral Blood Mononuclear Cells (PBMC), and eventually their synergistic effect, can be demonstrated. Through these complementary techniques, we observed a high radio-resistance of Caco-2 within the range of 2 - 10 Gy of X-rays and an increased Caco-2 monolayer permeability when PBMCs were added. In particular, PBMC presence was found to be associated with the variation in the tight junction scaffold proteins expression.

We present a protocol to investigate the crosstalk between X-ray-irradiated Caco-2 and peripheral blood mononuclear cells (PBMC). The protocol starts with Caco-2 irradiation and set-up of the co-culture with PBMC; subsequently, trans-epithelial electrical resistance is measured regularly over 48 h and western blot performed in both Caco-2 and PBMC.

X 射线辐照 Caco-2 细胞与 PBMC 间串扰的共培养方法研究

The protocol adopted in this work aims at unraveling how X-rays perturb the functioning of the intestinal barrier, focusing on the interplay between ...

免疫与感染

Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases including cancer. However, it is challenging to clarify the complex mechanism of intercellular interactions in highly heterogeneous cell populations using conventional co-culture systems because the heterogeneity of the cell subpopulation is obscured by the average values; the conventional co-culture systems can only be used to describe the population signal, but are incapable of tracking individual cells behavior. Furthermore, conventional single-cell experimental methods have low efficiency in cell manipulation because of the Poisson distribution. Microfabricated devices are an emerging technology for single-cell studies because they can accurately manipulate single cells at high-throughput and can reduce sample and reagent consumption. Here, we describe the concept and application of a microfluidic chip for multiple single-cell co-cultures. The chip can efficiently capture multiple types of single cells in a culture chamber (~46%) and has a sufficient culture space useful to study the cells&#39; behavior (e.g., migration, proliferation, etc.) under cell-cell interaction at the single-cell level. Lymphatic endothelial cells and oral squamous cell carcinoma were used to perform a single-cell co-culture experiment on the microfluidic platform for live multiple single-cell interaction studies.

This report describes a microfluidic chip-based method to set up a single cell culture experiment in which high-efficiency pairing and microscopic analysis of multiple single cells can be achieved.

使用微流体芯片以确定性方式建立基于单细胞的共培养物

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases ...

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.

人类干细胞衍生内皮细胞和GABAergic神经元的迁移、化学吸引和共培养测定

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have ...

神经科学

<meta charset="utf8"></head><body>用于上皮细胞和成纤维细胞的先进双层 3D 共培养系统

Establishing a Three-Dimensional Coculture Module of Epithelial Cells Using Nanofibrous Membranes

Technical hurdles in a culture of epithelial cells include dedifferentiation and loss of function. Biomimetic three-dimensional (3D) cell culture methods can enhance cell culture efficiency. This study introduces an advanced two-layered culture system intended to cultivate epithelial cells as tissue-like layers with the culture of fibroblasts within a 3D environment. Polyvinyl alcohol (PVA) and poly(&#949;-caprolactone) (PCL) nanofibrous membranes (NMs) were fabricated via electrospinning and utilized as a physiologically relevant extracellular matrix for the culture of epithelial cells and fibroblasts, respectively. In the upper insert wells, lung epithelial cells were cultivated on the PVA NM, and in the lower chambers, fibroblasts were cultured on the PCL NM. This configuration eliminates direct cell-cell contact and facilitates the examination of paracrine signaling mediated by soluble factors. Confocal microscopy was employed to analyze the distribution, growth pattern, and expression of intracellular proteins, including zona occludens in epithelial cells. Z-stacking techniques enabled detailed 3D reconstructions, providing precise insights into the integrity of tight junctions and spatial organization within the epithelial layer. Scanning electron microscopy (SEM) assessed the morphological characteristics of cell types on the nanofibrous membranes. SEM imaging revealed intricate cell surface structures and interactions with the nanofibers, offering a comprehensive perspective on cellular architecture and cell interaction with nanofibrous structure. The Cell Counting Kit-8 (CCK-8) assay is a simple method for measuring epithelial cell and fibroblast growth rates over time. It provides the proliferative behaviors and potential synergistic effects of coculturing these cells. These findings highlight the effectiveness of a simple insert co-culture system for simultaneous culture of fibroblasts and epithelial cells, which is crucial in various physiological and pharmacological contexts, including epithelial tissue regeneration, tumor microenvironment with endothelial, immune, and other stroma cells, toxicity assay, and drug activity test.

<meta charset="utf8"></head><body>作者聚焦:使用 PVA-PCL 纳米纤维膜推进 3D 共培养系统

Here, we demonstrate how epithelial cells cultured with fibroblasts in the nanofibrous membrane-based two-layer system can stably adhere to and grow on the membrane.

使用纳米纤维膜建立上皮细胞的三维共培养模块

Technical hurdles in a culture of epithelial cells include dedifferentiation and loss of function. Biomimetic three-dimensional (3D) cell culture methods ...

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

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

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

创新的 3D 打印插入物，旨在实现简单的 2D 和 3D 细胞培养

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

在细胞间接触缺失的两个蜂窝型的插入共培养系统的开发

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使用纳米纤维膜建立上皮细胞的三维共培养模块

使用纳米纤维膜建立上皮细胞的三维共培养模块

创新的 3D 打印插入物，旨在实现简单的 2D 和 3D 细胞培养