Name: a proximal culture method to study paracrine signaling between cells
Uploaded: 2018-08-28T14:30:00.000+00:00
Duration: 8 min 17 s
Description: Watch this Scientific Journal Video about A Proximal Culture Method to Study Paracrine Signaling Between Cells at JoVE.com

我们感谢患者参与这些实验的组织收集。国防部 OCRP 卵巢癌学院奖 (W81XWH-15-0253) 和一个试点奖从科琳的梦想基金会 Anirban k. 米特拉支持这项研究。

该议定书遵循印第安纳大学机构管理委员会的指导方针。
1. 细胞制剂
<ol><li>
人原发性间皮细胞的分离与培养
	<ol>
<li>从人网膜中分离人原发性间皮细胞 (HPMCs), 如前所述17、18和生长在完全生长培养基 [Dulbecco 的改良鹰的培养基 (DMEM) 含有10% 胎牛血清, 1%青霉素-链霉素, 1% 非必需氨基酸, 1% 维生素] 在37摄氏度和 5% CO2。 注: HPMCs 通常生长到100% 汇合处 (图 1A)。</li>
	</ol>
</li>
	<li>
卵巢癌细胞生长条件的研究
	<ol>
<li>生长 HeyA8 卵巢癌细胞在完全生长培养基在37°c 和 5% CO2。 注意: 使用 HeyA8 细胞生长到大约80% 汇合处 (图 1B)。</li>
	</ol>
</li>
</ol>2. 细胞培养
<ol><li>
近端区域性设置
	<ol>
<li>使用 Transwell 插入6井板与10微米厚的组织培养处理聚碳酸酯膜与0.4 微米毛孔 (108毛孔/cm2) 和4.67 厘米2生长区。如果需要, 可通过缩放根据生长表面积来播种的细胞数量来优化不同的插入尺寸。热完全生长培养基, 胰蛋白酶, 和磷酸盐缓冲盐水 (PBS) 到37摄氏度使用前。</li>
	</ol>
</li>
	<li>
准备插入
	<ol>
<li>要将插入膜下部表面的 HeyA8 细胞播种, 请使用无菌钳从包装中取出插入物, 并将其倒置在不育的15厘米培养皿中。</li>
		<li>使用15厘米的菜, 因为它有深度容纳倒置插入, 或替代任何适当的无菌容器。根据实验, 将所需的插入数放置在15厘米的盘子中。</li>
		<li>标签三控制和三实验条件相应地与标记在刀片的边缘。</li>
	</ol>
</li>
	<li>
准备癌细胞
	<ol>
<li>为了胰蛋白酶处理在25厘米2瓶 (~ 2 x 106细胞) 的80% 汇合处生长的 HeyA8 细胞, 添加1毫升的胰蛋白酶 (0.25%) 用于 40–60 s, 中和胰蛋白酶与6毫升完全生长培养基。</li>
		<li>将细胞以室温 (RT) 500 x g的速度离心成3分钟, 丢弃上清, 并在5毫升的完全生长培养基中并用重悬细胞颗粒。用台盼蓝染料排除法对活细胞进行计数。</li>
	</ol>
</li>
	<li>
培养癌细胞
	<ol>
<li>在完全生长培养基中悬浮10万细胞/毫升活 HeyA8 细胞。小心种子 8万 HeyA8 细胞悬浮在 800 ul 的完全生长培养基上的插入 (现在正朝上, 因为它是倒置)。</li>
		<li>种子单元格形成一个圆顶状形状, 使单元格保持在插入上 (图 1C)。从膜的中心开始, 在同心圆内向外移动, 慢慢吹打。避免超过3毫米从边缘, 以防止任何溢出的介质。 注: 种子细胞数量将取决于细胞的生长速率和实验的持续时间。对近端培养的 HeyA8 细胞数量进行了优化, 在第三天结束时, 细胞将80–90% 汇合。</li>
	</ol>
</li>
	<li>
肿瘤细胞附件
	<ol>
<li>盖上15厘米的盘子, 小心地将含有插入物的盘子移到 CO2孵化器中。在这个步骤中, 不中断插入的介质和单元格的放置非常关键。将细胞保持在37°c 4 小时, 使癌细胞附着在插入物上。</li>
	</ol>
</li>
	<li>
准备间皮细胞
	<ol>
<li>在大约 4 h 以后, 胰蛋白酶处理 HPMCs 生长在100% 汇合在 75 cm2瓶 (~ 4 x 106个细胞) 与2毫升胰蛋白酶 (0.25%) 为对的中最小和中和胰蛋白酶与12毫升完全生长培养基。</li>
		<li>离心细胞在 500 x g在 RT 3 分钟, 并用重悬细胞颗粒在10毫升的完全生长培养基, 并计数的活细胞使用台盼蓝染料排除方法。</li>
	</ol>
</li>
	<li>
将间皮细胞播种
	<ol>
<li>在完全生长培养基中悬浮30万细胞/毫升活 HPMCs。将2.5 毫升新鲜完整的生长介质添加到6井板的每一个井中。小心地把15厘米的盘子里装有插入物的生物安全罩。使用无菌钳, 翻转插入和放置在6井板的井, 使其直立, 与 HeyA8 细胞附着在较低的表面浸入完全生长介质 (图 1C)。</li>
		<li>种子 45万 HPMCs 悬浮在1.5 毫升的生长培养基内的插入与 HeyA8 细胞附着在表面上面对井底 (图 1C) 或插入没有任何 HeyA8 细胞的 HPMC 控制。添加1.5 毫升的生长培养基, 没有细胞的 HeyA8 控制。</li>
	</ol>
</li>
	<li>
成长的近端文化
	<ol>
<li>让近端文化生长在 CO2孵化器在37°c 和 5% CO2为 72 h。如有需要, 可按如下方式补充介质。<ol>
<li>对于内插入, 删除750µL 和添加750µL 新鲜完整的生长培养基。</li>
			<li>对于插入的外部 (6 井板), 去除1毫升和增加1毫升新鲜完全生长培养基。1毫升的选择是为了方便改变的媒介在一步。如果需要, 可以删除1.25 毫升, 取而代之。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
收集单元格
	<ol>
<li>在近端培养72小时后采集细胞。在这一步骤中特别小心, 以防止交叉污染的 HeyA8 细胞和 HPMCs. 胰蛋白酶处理细胞, 离心, 并溶解颗粒, 而不是株溶藻细胞膜上的细胞, 以防止交叉污染。</li>
	</ol>
</li>
	<li>
Trypsinizing 细胞
	<ol>
<li>用 1x PBS 冲洗插入件的两侧。</li>
		<li>将插入件转移到新井中, 加入胰蛋白酶。加入0.5 毫升的胰蛋白酶到内插入, 并添加2毫升胰蛋白酶到6井板, 以胰蛋白酶处理癌细胞附着在下部的插入。在37摄氏度孵育2分钟的盘子。</li>
	</ol>
</li>
	<li>
中和胰蛋白酶
	<ol>
<li>加入1毫升的完全生长培养基内的插入, 以中和的胰蛋白酶, 吸管向上和向下, 然后采取细胞和转移到一个标记的收集管。添加额外的2毫升完整的生长培养基, 完全中和。应注意不要在吹打时刺穿细胞膜, 以防止细胞交叉污染。</li>
		<li>要收集插入底部的单元格, 请将步骤12.2 中添加的2毫升胰蛋白酶移到下表面 2–3x, 使细胞落入6井板的底部。将该板块底部的胰蛋白酶与6毫升的生长培养基中和, 将该板块的内容转移到标记的收集管上。</li>
	</ol>
</li>
	<li>
收集和株溶藻细胞
	<ol>
<li>离心细胞在 500 x g 3 分钟, 并吸入介质。并用重悬和溶解以0.7 毫升苯酚-硫氰酸胍为基础的裂解试剂进行 RNA 提取。使用任何合适的工具包隔离 RNA。</li>
	</ol>
</li>
</ol>3. 定量实时聚合酶链反应
注: 以下步骤描述了定量的实时聚合酶链反应 (qPCR), 以研究基因表达的变化, 由于近端培养。
<ol><li>从 RNA 样品中制备 qPCR 的 cDNA。使用任何合适的逆转录酶与相应的缓冲和随机引物, 以确保所有 mRNA 的反向转录 (见材料表)。</li>
	<li>通过 qPCR 与甘油 3-磷酸脱氢酶 (GAPDH) 作为内部控制, 评估纤维连接蛋白 (FN1) 和 e-钙黏蛋白 (CDH1) mRNA 表达水平的变化和转化生长因子 beta 1 (TGFβ1)。可以使用标准化的商用基因表达化验 (见材料表) 或设计底漆。</li>
	<li>比较近端培养中的 HeyA8 细胞与 HPMCs 的 HeyA8 控制和近端培养的 HPMCs 与 HPMC 的对照。</li>
</ol>

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作者没有什么可透露的。

了解细胞间分泌和 juxtacrine 信号的机制对于培养对正常组织稳态和疾病状况的更好的认识是至关重要的,7,8。大多数分泌信号的研究是通过收集条件培养基从一个细胞类型和使用它来治疗其他细胞类型。这种方法在其固有的简单性方面具有优势。然而, 它并没有准确地重述细胞微环境中分泌因子的局部浓度, 或所涉及因素的产生和消耗的动力学。虽然前者可以在一定程度上解决, 虽然不准确, 通过集中的条件介质, 很难重新创造的动力学。使用 transwell 插入在井的插入和底部生长细胞, 在某种程度上, 可以重新捕获生产和损耗动力学的影响。然而, 这些细胞是由插入物和井底之间的距离隔开的, 因此无法重新创建分泌因子的局部浓度。我们报告了一个简单的近端培养方法研究相互分泌信号, 保留了局部高浓度的分泌因子, 以及生产率的动态和消耗的因素。
在一项原则验证的研究中, 我们证明了近距离培养方法在再现卵巢癌细胞与 HPMCs 相互作用的报告效果方面的有效性。此外, 我们还显示了相互分泌相互作用对 HeyA8 细胞和 HPMCs 上皮标记 e-钙黏蛋白表达的相反影响。虽然我们的实验涉及培养细胞在3天的时间, 它可以适应更短或更长的时间点通过调整初始细胞数量的种子。这使研究人员能够研究分泌信号的短期以及长期影响, 或进行时间课程研究。除了对肿瘤微环境的研究外, 这种简单的方法还可以应用于对两种黏附细胞类型之间的分泌信号的任何研究, 包括免疫学、血管生成、细胞发育和伤口愈合。
此方法的关键步骤是将细胞膜下部的细胞播种, 并从端点的两个曲面上收获细胞。我们通过先翻转插入, 然后将细胞播种成一大滴培养基, 在较低的表面上实现了播种。所研究的两个粘附细胞应在底面上播种。必须注意不要干扰插入, 而孵化它, 以避免这一斑点的媒介扰乱。硅胶可应用于刀片的边缘, 以避免液体气泡的破坏和溢出。在近端培养实验结束时, 膜两侧的细胞依次 trypsinized, 而不是裂解, 因为株溶藻膜上的细胞膜有可能通过毛孔交叉污染裂解物。通过将它们 trypsinizing 在各自的车厢内, 并注意到培养基和胰蛋白酶体积永远不会超过协议规定的数量, 因此避免了交叉污染。每个细胞类型收集在一个单独的管, 离心, 和细胞颗粒然后裂解。
虽然这种技术允许有效地研究分泌的相互作用, 同时避免公开的直接交互, 但我们不能排除通过隧道 nanotubules (TNTs) 的相互作用。这些 TNTs 中的一些可以很窄, 能够通过0.4 µm 毛孔, 长到足以跨越膜的10µm 厚度。虽然我们试图通过共聚焦显微镜来验证它们在毛孔中的存在是没有结论的 (数据没有显示), 但电子显微镜可能被用来证实它们的存在。因此, 如果候选细胞能够形成它们并通过它们进行交互, 则此方法不排除 TNTs 的潜在细胞通信。同样, 我们也不能排除由于细胞膜两侧的细胞可能堵塞毛孔而增加的分泌信号, 防止交叉扩散。
在0.4 µm 毛孔的膜的任何表面上生长的细胞也允许外来体的潜在交换。外来体被传统上认为是垃圾袋的细胞, 但最近的研究表明, 这些囊泡与他们的货物蛋白质, microRNAs, 甚至基因和 DNA 作为重要的调解人分泌蜂窝通讯22,23,24. 因此, 近端培养方法可以有效地用于通过外来体研究细胞通信。此外, 这种方法可以治疗与特定抑制剂的信号或中和抗体的分泌配体或相应的受体。总之, 我们报告了一个简单和多才多艺的近距离培养方法的发展, 它可以准确地复制分泌信号, 同时防止细胞之间的直接相互作用。这种方法的简便性使其广泛应用于癌症生物学、发展和免疫学等各个领域。

癌细胞与肿瘤微环境的相互作用在肿瘤进展中的作用已经得到了很好的确立, 并已成为癌症生物学研究的主要重点1。在伤口愈合、免疫应答、血管生成、干细胞龛位和发育过程中, 双向信号的相似情况是至关重要的,2、3、4、5、6,7,8. 在所有这些生物过程中, 一个共同的主题是细胞以各种方式对其微环境中的细胞外信号作出反应, 这决定了体细胞的命运、组织生理学和疾病进展。因此, 重点日益转向发展对这种细胞通讯所涉及的机制的更好的了解。大多数这种相互作用涉及细胞之间的分泌或 juxtacrine 信号。分泌信号涉及特定信号因子的分泌由一个细胞, 这是由相应的受体在附近的另一个细胞感知, 触发反应, 在它9,10, 而 juxtacrine信号要求两个细胞的细胞成分之间的直接接触涉及11,12。
这种信号是组织稳态以及肿瘤微环境中的重要组成部分。癌细胞从肿瘤基质细胞中的分泌和 juxtacrine 因子中受益, 包括肿瘤相关的成纤维细胞 (咖啡馆)、免疫细胞和脂肪瘤13、14、15、16。分泌信号可以由生长因子、细胞因子、趋化因子等介导, 而 juxtacrine 信号则包括在凹槽信号中并列配体和受体, 或整合与其各自的相互作用细胞外基质蛋白。我们已经证明了卵巢癌细胞与咖啡馆在肿瘤进展和转移中相互作用的重要性14。同样, 转移卵巢癌细胞与覆盖转移部位的间皮细胞的相互作用, 调节癌细胞的关键 microRNAs 和转录因子, 促进转移性殖民化17,18。
大多数关于分泌信号的研究涉及使用从一个细胞类型收集的条件培养基来治疗第二细胞类型。虽然这种方法已被广泛使用, 但它并没有有效地复制在接收细胞微环境中分泌因子的局部高浓度水平。它也无法重现由一个细胞产生的分泌因子连续流动的动力学, 相邻细胞接收。分泌信号是有效的短距离, 因为分泌因子是在所需浓度仅在源细胞附近, 并倾向于扩散和稀释, 随着距离的增加。这种局部高浓度的分泌因子对于触发受体细胞的反应至关重要。此外, 受体细胞的反应也依赖于新分泌因子的平衡, 以及它们在受体细胞中的退化、束缚和内化以及从源细胞扩散的持续损耗。条件培养基可以集中在微环境中存在的较高的局部浓度, 但不能准确地复制准确的浓度。此外, 它不能模仿生产的自然动力学和所涉及的因素的耗尽。为了更准确地复制分泌信号并将其与 juxtacrine 信号机制分开, 我们设计了一种新的近距离培养方法, 它涉及在多孔膜的任一表面上生长两种细胞类型。毛孔小到足以防止 juxtacrine 相互作用, 但允许在局部高浓度下交换分泌因子。这样, 系统就保留了生产的动力学和分泌因素的损耗。

<table><tbody><tr><td>24 mm Transwell permeable support with 0.4 &amp;micro;m Pore Polycarbonate Membrane Insert</td><td>Corning (Costar)</td><td>3412</td><td>&amp;bull; 10 &amp;micro;m thick translucent polycarbonate membrane&lt;br/&gt; &amp;bull; Treated for optimal cell attachment&lt;br/&gt; &amp;bull; Packaged 6 inserts in a 6 well plate, 4 plates per case&lt;br/&gt; &amp;bull; Membrane must be stained for cell visibility&lt;br/&gt; &amp;bull; Sterilized by gamma radiation</td></tr><tr><td>6 well plate</td><td>Corning (Falcon)</td><td>353046</td><td>Flat Bottom, TC-treated, sterile, with Lid</td></tr><tr><td>15 cm culture dish</td><td>Corning (Falcon)</td><td>353025</td><td>Sterile, TC-treated Cell Culture Dish</td></tr><tr><td>DMEM</td><td>Corning (Cellgro)</td><td>10-013-CV</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>Corning</td><td>30-002-CI</td><td></td></tr><tr><td>MEM Nonessential amino acids</td><td>Corning (Cellgro)</td><td>25-025-CI</td><td></td></tr><tr><td>MEM Vitamins</td><td>Corning (Cellgro)</td><td>25-020-CI</td><td></td></tr><tr><td>0.25% Trypsin, 2.21 mM EDTA</td><td>Corning</td><td>25-053-CI</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Atlanta Biologicals</td><td>S11150</td><td></td></tr><tr><td>Pipets</td><td></td><td></td><td>Any make is fine</td></tr><tr><td>CO2 Incubator</td><td></td><td></td><td>Any make is fine</td></tr><tr><td>Biosafety level II cabinet</td><td></td><td></td><td>Any make is fine</td></tr><tr><td>FN1 TaqMan Gene Expression Assay</td><td>ThermoFisher Scientific</td><td>Hs01549976_m1</td><td></td></tr><tr><td>TGFB1 TaqMan Gene Expression Assay</td><td>ThermoFisher Scientific</td><td>Hs00998133_m1</td><td></td></tr><tr><td>CDH1 TaqMan Gene Expression Assay</td><td>ThermoFisher Scientific</td><td>Hs01023895_m1</td><td></td></tr><tr><td>GAPDH TaqMan Gene Expression Assay</td><td>ThermoFisher Scientific</td><td>Hs99999905_m1</td><td></td></tr><tr><td>miRNeasy mini RNA isolation Kit</td><td>Qiagen</td><td>217004</td><td></td></tr><tr><td>High-Capacity cDNA Reverse Transcription Kit</td><td>ThermoFisher Scientific</td><td>43-688-13</td><td></td></tr><tr><td>HeyA8 ovarian cancer cells</td><td>Obtained from Ernst Lengyel Lab, University of Chicago</td><td></td><td></td></tr><tr><td>TGF&amp;beta; Neutralizing Antibody</td><td>R&amp;amp;D Systems</td><td>MAB1835-100</td><td></td></tr></tbody></table>

a proximal culture method to study paracrine signaling between cells

细胞间相互作用在许多生物学过程中起着重要作用, 包括肿瘤进展、免疫反应、血管生成和发育。分泌或 juxtacrine 信号介导这种相互作用。使用条件中等和共培养研究是区分这两种相互作用的最常见的方法。然而, 在分泌相互作用过程中, 局部高浓度分泌因子在微环境中的作用不是通过条件培养基准确概括的, 因此可能导致不精确的结论。为了克服这一问题, 我们设计了一种近端培养方法来研究分泌信号。两种细胞生长在10µm 厚的聚碳酸酯膜的任何表面, 0.4 µm 毛孔。毛孔允许交换分泌因子, 同时抑制 juxtacrine 信号。可以在端点收集和裂解细胞, 以确定分泌信号的影响。除了允许分泌因子的局部浓度梯度外, 这种方法还能经受长时间的培养以及抑制剂的使用的实验。利用这种方法研究卵巢癌细胞与转移部位间皮细胞的相互作用, 可以适应任何两种黏附细胞类型, 研究分泌信号在各个领域的应用,包括肿瘤微环境、免疫学和发育。

转移卵巢癌细胞在腹膜腔内的转移部位出现间皮细胞19。生产分泌和 juxtacrine 与间皮细胞的相互作用有助于诱导卵巢癌细胞的适应性反应, 从而成功转移17,18,20,21.为了检验近端培养方法的有效性, 我们测试了 HeyA8 卵巢癌细胞与 HPMCs 的分泌相互作用。以前有报道说, 共培养的 HeyA8 细胞与 HPMCs 导致了纤维粘连蛋白在 HPMCs21的表达增加。这种纤维粘连蛋白表达的诱导由 HeyA8 细胞的 TGFβ分泌增加介导21。因此, 我们测试了 HeyA8 细胞的近端培养和 HPMCs 对两种细胞纤维连接蛋白和 TGFβ的 mRNA 水平的影响。正如预期的那样, HeyA8 细胞的近端培养导致 HPMCs 中纤维粘连蛋白的表达增加, 与在没有 HeyA8 附着在下部的插入物的控制相比 (图 2A)。同样, HPMCs 与 HeyA8 细胞的近端培养导致 TGFβ在前者中的表达增加 (图 2B)。纤维连接蛋白和 TGFβ的表达在近端培养与 HPMCs 的 HeyA8 细胞中也显著增加, 与 HPMCs 未在插入物上表面播种的控制相比较 (图 2C和2D)。
为了比较经典条件培养基方法的效果, 我们对 HeyA8 细胞进行了实验治疗。纤维粘连蛋白表达减少, HeyA8 细胞 TGFβ的变化显著。(图 3A和3B)。我们还测试了在近端培养中用中和抗体阻断分泌 TGFβ的潜能。TGFβ中和抗体治疗后, HPMCs 近端培养 HeyA8 细胞 TGFβ的诱导明显减少 (图 3C)。然而, TGFβ中和抗体略微增加了 TGFβ在控制 HeyA8 细胞中的表达, 可能是作为补偿反应 (图 3C)。
此外, 我们还测试了近端培养对上皮标记 e-钙黏蛋白表达水平的影响。当 HeyA8 细胞的近端培养在 HPMCs (图 2E) 中略微增加 e-钙黏蛋白表达时, 在 HPMCs 附近生长的 HeyA8 细胞中观察到 e-钙黏蛋白的明显减少 (图 2F)。这些结果表明, 近端培养系统在研究卵巢癌细胞与转移部位正常细胞之间的分泌信号的有效性, 在这两种细胞类型中影响基因表达的转移性殖民化过程。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58144/58144fig1.jpg"> 图 1: 近端培养系统的装配.(A) 本小组显示人主要间皮细胞 (HPMCs)。(B) 本小组显示 HeyA8 卵巢癌细胞。刻度条 = 200 µm (A B)。(C) HeyA8 细胞被播种在 transwell 插入物的下表面, 有0.4 µm 毛孔。一旦细胞被附着, 插入物被放置在一个含有生长介质的6井板的井中。然后将间皮细胞播种在插入物上, 使它们附着在上表面, 并由细胞膜与 HeyA8 细胞分离。膜中的0.4 µm 毛孔允许分泌因子的交换, 但抑制细胞间的任何直接接触。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58144/58144fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58144/58144fig2.jpg"> 图 2: 近端培养对 mRNA 表达的影响.前两个面板显示的 qPCR (A) 纤维连接蛋白和 (B) TGFβ1表达的人主要间皮细胞 (HPMCs) 培养下部的 HeyA8 细胞, 与控制 HPMCs 相比。接下来的两个面板显示 qPCR (C) 纤维连接蛋白和 (D) TGFβ1表达在 HeyA8 细胞培养下部 HPMCs, 与控制 Heya8 细胞相比。最后两个面板显示 qPCR 的 e-钙黏蛋白表达 (e) HPMCs 培养下部 HeyA8 细胞和 (F) HeyA8 细胞培养下部 HPMCs. N = 3, * p < 0.01。误差线表示三重复的标准偏差。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58144/58144fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58144/58144fig3.jpg"> 图 3: 人原发性间皮细胞 (HPMC) 条件培养基治疗对 HeyA8 细胞 mRNA 表达的影响.前两个面板显示的 qPCR (A) 纤维连接蛋白和 (B) TGFβ1表达 HeyA8 细胞。(C) 这最后一个小组显示了抑制分泌的 TGFβ1与中和抗体 (TGFβ1 Ab) 对 HeyA8 TGFβ1表达后的近端培养与 HPMCs 的效果. N = 3, * p < 0.01。** < 0.05。误差线表示三重复的标准偏差。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58144/58144fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about A Proximal Culture Method to Study Paracrine Signaling Between Cells at JoVE.com

A Proximal Culture Method to Study Paracrine Signaling Between Cells

分泌和 juxtacrine 细胞相互作用在许多生物学过程中起着重要作用, 包括肿瘤的进展、免疫反应、血管生成和发育。在这里, 一种近端培养方法用于研究分泌信号, 其中的分泌因子的局部浓度保持, 而防止直接细胞接触。

一种研究细胞间分泌信号的近距离培养方法

Intercellular interactions play an important role in many biological processes, including tumor progression, immune responses, angiogenesis, and ...

a-proximal-culture-method-to-study-paracrine-signaling-between-cells

Universidad San Sebastian

Research

JoVE Journal

Biology

9.4K 次观看.  Indiana University School of Medicine. 分泌和 juxtacrine 细胞相互作用在许多生物学过程中起着重要作用, 包括肿瘤的进展、免疫反应、血管生成和发育。在这里, 一种近端培养方法用于研究分泌信号, 其中的分泌因子的局部浓度保持, 而防止直接细胞接触。

视频: A Proximal Culture Method to Study Paracrine Signaling Between Cells

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

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor environment and cell-cell interactions. Identification of cancer and stem cell interactions, including changes in extracellular environment, physical interactions, and secreted factors, might enable the discovery of new therapy options. We combine known co-culture techniques to create a model system for mesenchymal stem cells (MSCs) and cancer cell interactions. In the current study, dental pulp stem cells (DPSCs) and PC-3 prostate cancer cell interactions were examined by direct and indirect co-culture techniques. Condition medium (CM) obtained from DPSCs and 0.4 &#181;m pore sized trans-well membranes were used to study paracrine activity. Co-culture of different cell types together was performed to study direct cell-cell interaction. The results revealed that CM increased cell proliferation and decreased apoptosis in prostate cancer cell cultures. Both CM and trans-well system increased cell migration capacity of PC-3 cells. Cells stained with different membrane dyes were seeded into the same culture vessels, and DPSCs participated in a self-organized structure with PC-3 cells under this direct co-culture condition. Overall, the results indicated that co-culture techniques could be useful for cancer and MSC interactions as a model system.

We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction.

骨髓间充质干细胞在浆组织中的分离及与癌细胞联合培养研究其相互作用

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer growth and metastasis. Paracrine signals can involve cell-cell contact between different cell types, such as PD-L1 expressed on the surface of tumors interacting directly with PD-1 on the surface of T cells, or the secretion of ligands by a tumor cell to affect an immune cell. Here we describe a co-culture method to interrogate the effects of tumor-secreted ligands on immune cell (macrophage) activation. This straightforward procedure utilizes commercially available 0.4 &#181;m polycarbonate membrane permeable supports and standard tissue culture plates. In the process described, macrophages are cultured in the upper chamber and tumor cells in the lower chamber. The presence of the 0.4 &#181;m barrier allows for the study of intercellular signaling without the confounding variable of physical contact, because the two cell types share the same medium and exposure to paracrine ligands. This approach can be combined with others, such as genetic alterations of the macrophage (e.g., isolation from genetic knock-out mice) or tumor (e.g., CRISPR-mediated alterations) to study the role of specific secreted factors and receptors. The approach also lends itself to standard molecular biological analyses such as quantitative reverse transcription polymerase chain reaction (qRT-PCR) or Western blot analysis, without the need for flow sorting to separate the two cell populations. Enzyme-linked immunosorbent assays (ELISAs) can similarly be utilized to measure secreted ligands to better understand the dynamic interaction of cell signaling in the multiple cell type context. Duration of co-culture can also be varied for the study of temporally regulated events. This co-culture method is a robust tool that facilitates the study of tumor-secreted signals in the immune context.

Here, we present a method using permeable membrane supports to facilitate the study of non-contact paracrine signaling used by tumor cells to suppress the immune response. The system is amenable to studying the role of tumor-secreted factors in dampening macrophage activation.

研究肿瘤分泌性副体结扎对宏噬细胞活化的影响，使用可渗透膜支架共培养

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer ...

A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. Without sensory innervation, daily oral activities would cause irreparable damage. Despite its importance, the roles of innervation in tooth development and maintenance have been largely overlooked. Several studies have demonstrated that DP cells secrete extracellular matrix proteins and paracrine signals to attract and guide TG axons into and throughout the tooth. However, few studies have provided detailed insight into the crosstalk between the DP mesenchyme and neuronal afferents. To address this gap in knowledge, researchers have begun to utilize co-cultures and a variety of techniques to investigate these interactions. Here, we demonstrate the multiple steps involved in co-culturing primary DP cells with TG neurons dispersed on an overlying transwell filter with large diameter pores to allow axonal growth through the pores. Primary DP cells with the gene of interest flanked by loxP sites were utilized to facilitate gene deletion using an Adenovirus-Cre-GFP recombinase system. Using TG neurons from the Thy1-YFP mouse allowed for precise afferent imaging, with expression well above background levels by confocal microscopy. The DP responses can be investigated via protein or RNA collection and analysis, or alternatively, through immunofluorescent staining of DP cells plated on removable glass coverslips. Media can be analyzed using techniques such as proteomic analyses, although this will require albumin depletion due to the presence of fetal bovine serum in the media. This protocol provides a simple method that can be manipulated to study the morphological, genetic, and cytoskeletal responses of TG neurons and DP cells in response to the controlled environment of a co-culture assay.

We describe the isolation, dispersion and plating of dental pulp (DP) primary cells with trigeminal (TG) neurons cultured atop overlying transwell filters. Cellular responses of DP cells can be analyzed with immunofluorescence or RNA/protein analysis. Immunofluorescence of neuronal markers with confocal microscopy permits the analysis of neurite outgrowth responses.

研究对牙髓寄生虫信号反应的神经质生长的共同培养方法

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. ...

发育生物学

Modeling Paracrine Noncanonical Wnt Signaling In Vitro

Noncanonical Wnt signaling regulates intracellular actin filament organization and polarized migration of progenitor cells during embryogenesis. This process requires complex and coordinated paracrine interactions between signal-sending and signal-receiving cells. Given that these interactions can occur between various types of cells from different lineages, in vivo evaluation of cell-specific defects can be challenging. The present study describes a highly reproducible method to evaluate paracrine noncanonical Wnt signaling in vitro. This protocol was designed with the ability to (1) conduct functional and molecular assessments of noncanonical Wnt signaling between any two cell types of interest; (2) dissect the role of signal-sending versus signal-receiving molecules in the noncanonical Wnt signaling pathway; and (3) perform phenotypic rescue experiments with standard molecular or pharmacologic approaches.
This protocol was used to evaluate neural crest cell (NCC)-mediated noncanonical Wnt signaling in myoblasts. The presence of NCCs is associated with an increased number of phalloidin-positive cytoplasmic filopodia and lamellipodia in myoblasts and improved myoblast migration in a wound-healing assay. The Wnt5a-ROR2 axis was identified as a crucial noncanonical Wnt signaling pathway between NCC and second heart field (SHF) cardiomyoblast progenitors. In conclusion, this is a highly tractable protocol to study paracrine noncanonical Wnt signaling mechanisms in vitro.

Modeling Paracrine Noncanonical Wnt Signaling In Vitro

The present study outlines a highly reproducible and tractable method to study paracrine noncanonical Wnt signaling events in vitro. This protocol was applied to evaluate the impact of paracrine Wnt5a signaling in murine neural crest cells and myoblasts.

体外模拟旁非规范Wnt信号传导

Noncanonical Wnt signaling regulates intracellular actin filament organization and polarized migration of progenitor cells during embryogenesis. This ...

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

A Cell Culture Model of Resistance Arteries

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and signaling processes that can control vascular tone and blood pressure. As it is a projection from either the endothelial or smooth muscle cell, and due to its small size (on average, an area of ~1 &#181;m2), the MEJ is difficult to study in isolation. However, we have developed a cell culture model called the vascular cell co-culture (VCCC) that allows for in vitro MEJ formation, endothelial cell polarization, and dissection of signaling proteins and processes in the vascular wall of resistance arteries. The VCCC has a multitude of applications and can be adapted to suit different cell types. The model consists of two cell types grown on opposite sides of a filter with 0.4 &#181;m pores in which the in vitro MEJs can form. Here we describe how to create the VCCC via plating of cells and isolation of endothelial, MEJ, and smooth muscle fractions, which can then be used for protein isolation or activity assays. The filter with intact cell layers can be fixed, embedded, and sectioned for immunofluorescent analysis. Importantly, many of the discoveries from this model have been confirmed using intact resistance arteries, underscoring its physiological relevance.

A cell culture model of resistance arteries is described, allowing for the dissection of signaling pathways in endothelium, smooth muscle, or between endothelium and smooth muscle (the myoendothelial junction). The selective application of agonists or protein isolation, electron microscopy, or immunofluorescence can be utilized using this cell culture model.

阻力动脉的细胞培养模型

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and ...

医学

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

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

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

创新的 3D 打印插入物，旨在实现简单的 2D 和 3D 细胞培养

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

生物学

3D Co-culture with Direct Interaction: Co-culturing Cancer Cells with Monocytes to Study Their Interaction

Source: Espinoza-S&#225;nchez, et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/56589/analyzing-communication-between-monocytes-primary-breast-cancer-cells">Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract (ECME)-based Three-dimensional System.</a> J. Vis. Exp. (2018).
This video describes the 3D co-culture of breast cancer cells with monocytes to study their interaction in an extracellular matrix extract (ECME) based environment. This method helps in studying specific features such as collagen degradation, immune cell recruitment, and cell invasion.

直接相互作用的 3D 共培养:癌细胞与单核细胞共培养以研究它们的相互作用

Source: Espinoza-S&#225;nchez, et al. Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ...

JoVE Encyclopedia of Experiments

乳腺癌

In vitro studies

Proximal Culture System: An In Vitro Culture Technique to  Study Paracrine Signaling Between Cells

Source: Dasari, S. et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/t/58144">A Proximal Culture Method to Study Paracrine Signaling Between Cells.</a>&#160;J. Vis. Exp. (2018)
In this video, we develop a proximal co-culture model of ovarian cancer cells and primary human mesothelial cells. This physiologically relevant model allows the study of paracrine signaling, where the localized concentrations of the secreted factors are maintained while preventing direct cellular contact.

近端培养系统:一种研究细胞间旁分泌信号传导的体外培养技术

Source: Dasari, S. et al. A Proximal Culture Method to Study Paracrine Signaling Between Cells.&#160;J. Vis. Exp. (2018)
In this video, we develop a ...