Name: cell patterning using magnetic-archimedes strategy
Uploaded: 2024-02-02T14:30:00.000+00:00
Duration: 5 min 9 s
Description: Watch this Scientific Journal Video about Magnetic-Based Cell Patterning Method for High-Throughput Biomedical Applications at JoVE.com

本研究得到了国家重点研发计划（2021YFA1101100）、国家自然科学基金（32000971）、中央高校基本科研业务费（2021FZZX001-42）和浙江大学上海高等研究院星夜科学基金（批准号：Grant No.SN-ZJU-SIAS-004）。

1. 组装磁铁组
<ol><li>组装条形图案的磁铁组。<ol><li>选择扁平矩形磁铁，如 图1A所示。用于此演示的矩形磁铁的尺寸为 1.5 mm × 10 mm × 35 mm（厚度×高度×长度）（参见 材料表）。磁铁的厚度决定了细胞条纹之间的间隙。</li>
		<li>将 2 毫米厚的硅胶板（参见 材料表）切成 2 毫米× 8 毫米× 30 毫米的矩形。确保这些硅胶板的后两个尺寸略小于上述矩形磁铁的尺寸，以优化组装。硅胶板的厚度决定了电池条纹的宽度。</li>
		<li>逐层组装矩形磁铁和硅胶板，如 图 1A 所示。每个磁铁组由 4 个磁铁和 3 个硅胶板组成。确保每个相邻磁铁的磁极相对，以便磁铁组可以由于内在吸引力而自动对齐。</li>
		<li>使用前对磁铁进行消毒。 注意：建议用纯水清洗磁铁组，然后将它们浸入 75% 乙醇中 10 分钟。这些磁体组的整体尺寸为 12 mm × 10 mm × 35 mm，设计用于标准 6 孔细胞培养板。</li>
	</ol></li>
	<li>组装点阵图案的磁铁组<ol><li>选择微型圆柱形磁铁，如 图1B所示。用于此演示的圆柱形磁铁的尺寸为 Φ1.5 mm × 10 mm（直径×高度），沿轴向磁化。</li>
		<li>如 图 1B 所示组装圆柱形磁铁。确保每个磁铁组由 36 个圆柱形磁铁组成，这些磁铁排列在 6 × 6 网格中。此外，确保每个相邻磁铁的磁极相对，以允许磁铁组由于内在吸引力而自动对齐。</li>
		<li>使用前对磁铁进行消毒，遵循与步骤 1.1.4 相同的步骤。这些磁体组的整体尺寸为 9 mm × 10 mm × 9 mm，设计用于标准 6 孔细胞培养板。</li>
	</ol></li>
</ol>2. 载玻片上的细胞图案
<ol><li>准备细胞培养装置。<ol><li>使用 2 毫米厚的硅胶板制作硅胶模具。使用打孔器在板上创建一个 1 厘米× 1 厘米的矩形孔。在孔周围修剪硅胶板，以制作一个圆形模具（直径 = 25 mm），孔位于中心，如 图 1C 所示。</li>
		<li>使用超声波清洗机彻底清洁硅胶模具。通过用纯净水洗涤模具10分钟来灭菌模具，然后用75%乙醇代替水再消毒10分钟。最后，将模具在65°C的灭菌器盒中干燥60分钟。</li>
		<li>将灭菌的硅胶模具连接到Φ25 mm圆形玻璃细胞载玻片上，轻轻按压，使硅胶粘附在玻璃上，如 图1C所示。由此产生的细胞培养装置将具有玻璃底面和 200 μL 培养腔。该装置的尺寸为 Φ25 mm × 2.15 mm，适用于标准 6 孔细胞培养板。 注意：上述细胞培养装置可以用其他底面薄于 0.5 毫米的培养皿代替，例如共聚焦培养皿。</li>
		<li>将磁铁组放在 6 孔板上。建议使用非磁性镊子，以便于操作。 注意：从这一点开始，所有后续步骤都应在无菌条件下进行，直到观察到细胞模式。</li>
		<li>将细胞培养装置放置在 6 孔板孔中的磁铁组顶部。确保细胞培养装置水平放置，底部与磁铁组紧密接触。</li>
	</ol></li>
	<li>制备含有Gd-DTPA的细胞培养基<ol><li>制备市售的Gd-DTPA注射液（见 材料表），通常浓度为500mM。通过将 30 μL Gd-DTPA 注射液加入 470 μL 完全细胞培养基中来稀释进样液，以达到 30 mM 的目标浓度。 注：根据当地法规和可用性，具有相同目标浓度的其他钆基造影剂 （GBCA） 可能会产生类似的结果11.</li>
	</ol></li>
	<li>创建单元格模式。<ol><li>收获细胞进行图案化。在本演示中，使用人脐静脉内皮细胞（HUVEC，见 材料表）进行图案化（图2）。将细胞在200× g （室温下）离心5分钟，以从悬浮液中收集它们，并将它们重悬于含有Gd-DTPA的培养基中。计算细胞密度。</li>
		<li>用含 Gd-DTPA 的培养基将细胞密度调节至约 ~2 ×10 个 5 个 细胞/mL。轻轻混合细胞悬液，并向每个细胞培养模具的腔中加入 200 μL，以形成充满边缘的液体表面。</li>
		<li>小心地将板转移到细胞培养箱中并孵育3-6小时，直到细胞很好地粘附在底物上。避免砰的一声关上培养箱门，以防止干扰细胞图案的组装。对于粘附率低的细胞，用GBCAs过夜处理通常是安全的12。</li>
		<li>当细胞粘附在细胞培养装置的腔底部时，细胞图案化被认为是完整的。用完全培养基替换含Gd-DTPA的培养基。</li>
		<li>从磁铁组中取出细胞培养装置。人们可以立即观察细胞模式或将设备转移到新的培养板中进行进一步培养。</li>
	</ol></li>
</ol>3. 磁铁侧向共培养图案：移动模板的制作
注意：以下程序旨在利用基于 Mag-Arch 的单元图案并探索更多应用的可能性。
<ol><li>按照步骤 1 和步骤 2 准备设备以进行条纹单元图案化。但是，将细胞密度降低到1×105 个细胞/mL，并用更薄的硅胶板替换将磁铁分开的硅胶板，使每个条纹细胞图案更薄。 注意：这为以条纹模式放置多个单元格提供了足够的区域。作为参考，我们建议使用 1 毫米硅胶板而不是 2 毫米来分离磁铁。</li>
	<li>对第一种类型的单元格进行图案化，如步骤 2.2-2.3 所示。此步骤还需要 3-6 小时进行细胞附着。 注意：为了区分共培养系统中的不同细胞类型，用户可以在图案化之前使用荧光染料（如 DiI、DiD 或细胞跟踪器（CMTPX、CMFDA、CMAC 等）标记细胞。例如，对于 DiI 和 DiD 染色，每 1 mL 无 FBS 培养基中加入 1 μL 储备溶液 （10 mM）（参见 材料表），并充分混合以获得工作溶液。用工作溶液替换培养基以覆盖贴壁细胞。孵育 30 分钟并用 PBS 洗涤 3 次后，继续进行细胞收获。</li>
	<li>对第一种类型的细胞进行图案化后，将细胞培养装置从下面的磁铁向右移动 1 mm，如 图 3Aii 所示。</li>
	<li>用 200 μL PBS 轻轻洗涤载玻片两次。避免让载玻片干燥。</li>
	<li>以步骤2.2-2.3中所示的相同方式对第二种类型的单元格进行图案化。</li>
	<li>如 图3Aiii所示，将细胞培养装置从下面的磁铁向右移动1 mm，并再次用200μLPBS洗涤载玻片。以步骤2.2-2.3中所示的相同方式对第三种类型的单元格进行图案化。</li>
	<li>对所有类型的细胞进行图案化后，用 200 μL PBS 轻轻洗涤载玻片两次，并用完整的细胞培养基替换。</li>
	<li>从磁铁组中取出细胞培养装置。然后可以观察细胞模式或将设备转移到新的培养板上进行进一步培养。</li>
</ol>4. 通过调节Gd-DTPA浓度进行共培养图案化
注意：GBCA在工作浓度（≤75 mM）下不会显着影响细胞粘附或随后的生长。此外，细胞模式受 Gd-DTPA 浓度的影响：浓度越高，细胞模式越小/越薄。因此，可以通过简单地调整Gd-DTPA的浓度来创建共培养系统。此示例演示了同心圆阵列的图案化。
<ol><li>使用步骤1.2中的微型圆柱形磁铁对第一种类型的电池进行图案化，如步骤2.2-2.3所示。在收获细胞之前，用荧光染料（如 DiI、DiD 或细胞追踪器）标记细胞，以区分共培养系统中的不同细胞类型。 注意：需要更高浓度的 Gd-DTPA（50 mM 而不是 30 mM）和更低的细胞密度，大约 1 × 10 个5 个 细胞/mL。</li>
	<li>对第一个细胞阵列进行图案化后，用 200 μL PBS 轻轻洗涤载玻片两次。避免让载玻片变干。</li>
	<li>按照与以前相同的步骤对第二种类型的细胞进行图案化。使载玻片在磁铁上保持相对不动，以防止脱位。建议将带有空心的硅胶板连接到载玻片的下表面，该空心适合磁铁组。 注意：在这里，需要较低浓度的Gd-DTPA（20mM而不是30mM），以便第二个细胞模式预计比第一个细胞模式大，如 图3B所示。</li>
	<li>对所有类型的细胞进行图案化后，用 200 μL PBS 轻轻洗涤载玻片两次，并用完全细胞培养基替换培养基。</li>
	<li>从磁铁组中取出细胞培养装置。然后可以观察细胞模式或将设备转移到新的培养板上进行进一步培养。</li>
</ol>5. 微流控芯片中的细胞图案化
注意：在我们之前的研究8 中，基于 Mag-Arch 的方法已被证明可以在封闭的狭窄腔室中工作。这是在微流体通道中图案化点阵的示例。
<ol><li>微流体芯片的制造<ol><li>定制 40 mm × 75 mm × 20 mm 聚四氟乙烯 （PTFE） 模具（参见 材料表），其中包含 24 mm × 50 mm × 8 mm 的矩形型腔，如 图 4A 所示。</li>
		<li>压平 0.5 毫米厚的硅胶板。根据流体通道的形状切割硅胶板，流体通道具有 15 mm × 20 mm × 0.5 mm 的矩形腔体，入口和出口侧都有三角形缓冲区，如图 4A，B 所示。</li>
		<li>定制 40 毫米× 75 毫米矩形玻璃板。用空气等离子清洗玻璃板 30 秒。</li>
		<li>空气等离子体清洗后，立即用 0.5 mL 市售抗粘连缓冲液覆盖玻璃板（参见 材料表）并让它们风干。用乙醇清洗玻璃板一次，然后让它们风干。</li>
		<li>将步骤5.1.2中制备的硅胶板牢固地连接到玻璃板的中间，如 图4A所示。</li>
		<li>根据制造商的说明制备聚二甲基硅氧烷（PDMS）预聚物（见 材料表）。对于每个PDMS芯片，将10 g碱成分和1 g固化剂称取到50 mL离心管中。</li>
		<li>用玻璃搅拌棒充分混合药剂，直到小气泡均匀分布在胶体中。混合需要5-10分钟，具体取决于胶体体积。将胶体预聚物在500× g （室温下）离心1分钟以消除气泡。</li>
		<li>将 ~10 mL 的预聚物缓慢倒入型腔中以填充 PTFE 模具。</li>
		<li>小心地将模具放入真空储液罐中并保持水平，以防止预聚物流走。</li>
		<li>用真空泵去除预聚物中残留的气泡。吸尘需要 120-180 分钟。</li>
		<li>如果需要，倒入更多的预聚物以进一步填充模具型腔。再吸尘 60 分钟。</li>
		<li>小心地用玻璃板盖住PTFE模具，二氧化硅面朝向型腔，如 图4Bi所示。</li>
		<li>确保消除所有气泡。将模具转移到60°C干燥箱中，使预聚物凝固过夜。</li>
		<li>从烤箱中取出模具。冷却后，小心地对凝固的PDMS盖进行脱模。用 Φ1 mm 针刺器创建入口和出口。</li>
		<li>按照步骤 2.1.2 清洗 PDMS 盖和 24 mm × 50 mm 盖玻片。 注意： 在无菌条件下继续执行以下步骤。</li>
		<li>连接PDMS盖和盖玻片，以创建包含高度约为~500μm的流道的微流体芯片。底部应由一个 ~0.15 mm 的玻璃盖玻片组成，以便使用基于 Mag-Arch 的策略进行细胞图案化。</li>
	</ol></li>
	<li>将市售的无菌适配器组装到微流体芯片8 的入口和出口中。</li>
	<li>制备2%（w / v，溶解在PBS中）明胶包衣缓冲液。通过高压灭菌对缓冲液进行灭菌。</li>
	<li>使用 1 mL 注射器 通过 入口适配器将明胶包被缓冲液注入芯片中。如果出现气泡，请用额外的涂层缓冲液将其冲洗掉。</li>
	<li>将芯片放入 10 cm 细胞培养皿中。在培养皿底部周围加入 1 mL PBS，以避免干燥。将培养皿转移到细胞培养箱中。涂层需要 30 分钟。</li>
	<li>通过入口用 2 mL 含 Gd 的培养基 （30 mM） 冲洗包被缓冲液。</li>
	<li>用 1 mL 注射器轻轻注射含有 30 mM Gd-DTPA 的 1 mL 细胞悬液。</li>
	<li>如步骤2.2-2.3所示的图案电池使用步骤1.2中的微型圆柱形磁铁。 注意： 但是，建议将带有适合磁铁组的空心硅胶板连接到载玻片的下表面，如 图 4Ci 所示。</li>
	<li>图案化后，用 1 mL 注射器冲洗，用 1 mL 完全培养基轻轻刷新含有 Gd-DTPA 的培养基。 注意：从步骤5.7-5.9开始，微流体中的细胞图案化过程大约需要180分钟，这类似于标准载玻片上的细胞图案化。</li>
	<li>在显微镜下量化图案。如果需要，将微流体芯片连接到循环培养装置以进行进一步培养。 注意：根据我们的经验，当由基于微泵的简单设备支持时，细胞能够生长至少 72 小时，该设备在细胞培养箱8 中为微流体提供连续流动的新鲜培养基。</li>
</ol>

一种新型的无墨水、无标记磁性阿基米德细胞图案化方法

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Levitational+3D+bioassembly+and+density-based+spatial+coding+of+levitoids.">Levitational 3D bioassembly and density-based spatial coding of levitoids.</a> Adv Funct Mater. 32 (50), 2204092 (2022).</li><li>Parfenov, V. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+levitational+bioassembly+of+3D+tissue+construct+in+space.">Magnetic levitational bioassembly of 3D tissue construct in space.</a> Sci Adv. 6 (29), eaba4174 (2020).</li><li>Dell, A. C., Wagner, G., Own, J., Geibel, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+bioprinting+using+hydrogels:+Cell+inks+and+tissue+engineering+applications.">3D bioprinting using hydrogels: Cell inks and tissue engineering applications.</a> Pharmaceutics. 14 (12), 2596 (2022).</li><li>Ino, K., Ito, A., Honda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+patterning+using+magnetite+nanoparticles+and+magnetic+force.">Cell patterning using magnetite nanoparticles and magnetic force.</a> Biotechnol Bioeng. 97 (5), 1309-1317 (2007).</li><li>Okochi, M., Matsumura, T., Honda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+force-based+cell+patterning+for+evaluation+of+the+effect+of+stromal+fibroblasts+on+invasive+capacity+in+3d+cultures.">Magnetic force-based cell patterning for evaluation of the effect of stromal fibroblasts on invasive capacity in 3d cultures.</a> Biosens Bioelectron. 42, 300-307 (2013).</li><li>Mishriki, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+magnetic+3D+printing+of+cellular+structures+with+mcf-7+cell+inks.">Rapid magnetic 3D printing of cellular structures with mcf-7 cell inks.</a> Research (Wash D C). 2019, 9854593 (2019).</li><li>Ozturk-Oncel, M. O., Leal-Martinez, B. H., Monteiro, R. F., Gomes, M. E., Domingues, R. M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dive+into+the+bath:+Embedded+3D+bioprinting+of+freeform+in+vitro+models.">A dive into the bath: Embedded 3D bioprinting of freeform in vitro models.</a> Biomater Sci. 11, 5462-5473 (2023).</li><li>Sahni, G., Yuan, J., Toh, Y. 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基于 Mag-Arch 的细胞图案化为大多数生物医学实验室提供了用户友好的解决方案。该方法与无墨水、无标记、独立于基材的特征以及高通量图案化的能力并行 8,13。对于单体细胞图案化，它以一步方式对细胞进行图案化。该过程只需刷新培养基即可完成。
以前的研究使用磁性粒子来标记细胞并用磁铁吸引它们以形成精确的图案<sup clas...

细胞图案化正在发展成为一种直观而强大的 体外 研究技术1.通过操纵培养板中的细胞位置，它为各种实验提供了解决方案，包括细胞迁移2、仿生多细胞共培养3、类器官组装4、生物材料研究5 等。在大多数情况下，无墨水、无标记方法是细胞图案化的首选方法，因为它为后续研究提供了易于操作和高细胞活力。
磁拱效应是一种物理现象，其中顺磁性液体中的抗磁性物体倾向于向磁场较弱的区域移动6.活细胞具有天然的抗磁性，而细胞培养基可以通过添加可溶性顺磁性元素制成顺磁性，例如钆喷酸二葡甲胺 （Gd-DTPA），通常用于静脉注射核磁共振成像作为造影剂7。因此，预计细胞将被周围的顺磁性介质排斥，并移动到磁场较弱的区域8.使用一组钕磁铁可以很容易地产生图案磁场。理想情况下，电池图案与磁铁图案相反。从技术上讲，这被定义为一种无标记方法，因为唯一的附加试剂 Gd-DTPA 保留在细胞外环境中并且不与细胞结合。因此，通过更换培养基，可以很容易地避免对后续细胞培养的潜在影响。与其他方法1,3,9,10相比，基于Mag-Arch的策略不需要生物墨水成分或应用特定颗粒来积极标记细胞。此外，它已被证明可以在多种底物上进行细胞粘附，并且能够实现高通量细胞图案化4。
本文介绍了使用基于 Mag-Arch 的方法进行细胞图案化的详细协议，涵盖了从设备制造到调整细胞图案的所有内容。除了我们已经演示的图案外，用户还可以使用磁铁和Gd-DTPA解决方案轻松创建各种单元图案。此外，还提供了用于在封闭的微流控芯片中组装复杂的共培养模式和操纵细胞的方案。

<table><tbody><tr><td>A2780 ovarian cancer cells</td><td>Procell</td><td>CL-0013</td><td></td></tr><tr><td>Cell culture medium (DMEM, high glucose)</td><td>Gibco</td><td>11995040</td><td></td></tr><tr><td>Cover slides</td><td>Citotest Scientific</td><td>80340-3610</td><td>For fabricating microfluidics. Dimension: 24 mm &amp;times; 50 mm</td></tr><tr><td>DiD</td><td>MedChemExpress (MCE)&amp;nbsp;</td><td>HY-D1028</td><td>For labeling cells with red fluorescence (Ex: 640 nm)</td></tr><tr><td>DiI</td><td>MedChemExpress (MCE)&amp;nbsp;</td><td>HY-D0083&amp;nbsp;</td><td>For labeling cells with orange fluorescence (Ex: 550 nm)</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Biochannel</td><td>BC-SE-FBS07</td><td></td></tr><tr><td>Gadopentetate dimeglumine (Gd-DTPA)</td><td>Beijing Beilu Pharmaceutical&amp;nbsp;</td><td>H10860002</td><td></td></tr><tr><td>Gelatin</td><td>Sigma Aldrich</td><td>V900863</td><td></td></tr><tr><td>Glass cell slides</td><td>Citotest Scientific</td><td>80346-2510</td><td>Diameter: 25 mm; thickness: 0.19-0.22 mm</td></tr><tr><td>Glass plates</td><td>PURESHI hardware store</td><td></td><td>For fabricating microfluidics. Dimension: 40 mm &amp;times; 75 mm</td></tr><tr><td>Human Umbilical Vein Endothelial Cells (HUVECs)</td><td>Servicebio</td><td>STCC12103G-1</td><td></td></tr><tr><td>Neodymium-iron-boron magnets (N52)</td><td>Lalaci</td><td></td><td></td></tr><tr><td>Non-toxic glass plate coating (Gel Slick Solution)</td><td>Lonza</td><td>1049286</td><td>For convenience of demolding when fabricating microfluidics</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Servicebio</td><td>G4200</td><td></td></tr><tr><td>Plasma cleaner</td><td>SANHOPTT</td><td>PT-2S</td><td></td></tr><tr><td>Polydimethylsiloxane (PDMS) kit</td><td>DOWSIL</td><td>SYLGARD 184 Silicone Elastomer Kit</td><td>For fabricating microfluidics</td></tr><tr><td>Polytetrafluoroethylene (PFTE) mold</td><td>PURESHI hardware store</td><td></td><td>Customized online, for fabricating microfluidics</td></tr><tr><td>Silicon plate</td><td>PURESHI hardware store</td><td></td><td></td></tr><tr><td>Smooth Muscle Cells (SMC)</td><td>Procell</td><td>CL-0517</td><td></td></tr><tr><td>Ultrasonic cleaner</td><td>Sapeen</td><td>CSA-02</td><td></td></tr></tbody></table>

cell patterning using magnetic-archimedes strategy

细胞图案化可以精确控制细胞定位，在细胞行为研究中具有独特的优势。在该协议中，引入了一种基于磁阿基米德（Mag-Arch）效应的细胞图案化策略。这种方法可以在不使用油墨材料或标记颗粒的情况下精确控制细胞分布。通过引入顺磁性试剂来增强细胞培养基的磁化率，细胞被磁铁排斥，并排列成与位于微流体基板下方的磁铁组互补的图案。
在本文中，提供了使用基于 Mag-Arch 的策略进行细胞图案化的详细过程。提供了用于单细胞类型以及用于共培养实验的多种细胞类型的图案化方法。此外，还提供了制造包含细胞图案化通道的微流控装置的综合说明。使用并行方法实现此功能具有挑战性，但可以以简化且经济高效的方式完成。采用基于 Mag-Arch 的细胞图案化为研究人员提供了强大的 体外 研究工具。

选择矩形（1.5 mm × 10 mm × 35 mm）和圆柱形（Φ1.5 m × 10 mm）磁体来创建电池图案作为演示。用户可以灵活地修改磁铁的尺寸和形状，或以不同的方式组装它们以创建不同的细胞图案。在图1A，B中，磁铁被组装起来，磁极用蓝色（南）和红色（北）描绘，以清晰起见。在这种配置中，磁铁横向相互吸引并自行对齐，如图 2 所示。<strong class="xfig...

Watch this Scientific Journal Video about Magnetic-Based Cell Patterning Method for High-Throughput Biomedical Applications at JoVE.com

作者聚焦：用于高通量生物医学应用的基于磁性的细胞图案化方法 

该协议描述了一种基于磁阿基米德效应的无墨水、无标记、独立于基板、高通量细胞图案化的方法。

使用磁阿基米德策略的细胞图案化

Cell patterning, allowing precise control of cell positioning, presents a unique advantage in the study of cell behavior. In this protocol, a cell ...

细胞图案化是用于体外研究的直观而强大的技术。我们的研究旨在提供一种多功能、用户友好且廉价的细胞模式方法，该方法可以在大多数生物医学实验室中复制。与其他方法相比，我们的协议不需要专门的技能和设备。此外，它还与无墨水、无标签、独立于基材的特征以及高通量图案化的能力并行。该协议只需要两种必需品：磁铁和钆基造影剂，它们都很容易以低廉的价格获得。基于磁性的细胞模式方法为各种实验的第一步提供了方便的解决方案，包括细胞迁移、仿生多细胞共培养、类器官组装、生物材料研究等。

cell-patterning-using-magnetic-archimedes-strategy

Research

JoVE Journal

Bioengineering

Cell Patterning Using Magnetic-Archimedes Strategy

1.3K 次观看.  Zhejiang University School of Medicine. 细胞图案化是用于体外研究的直观而强大的技术。我们的研究旨在提供一种多功能、用户友好且廉价的细胞模式方法，该方法可以在大多数生物医学实验室中复制。与其他方法相比，我们的协议不需要专门的技能和设备。此外，它还与无墨水、无标签、独立于基材的特征以及高通量图案化的能力并行。该协议只需要两种必需品：磁铁和钆基造影剂，它们都很容易以低廉?...

视频: 作者聚焦：用于高通量生物医学应用的基于磁性的细胞图案化方法 

Patterning of Embryonic Stem Cells Using the Bio Flip Chip

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such as tumor growth, stem cell differentiation, and stem cell self-renewal. A number of methods currently exist to modulate cell signaling in vitro. One method of modulating the total amount of diffusible signaling is to vary the cell seeding density during culture. Due to the random nature of cell seeding, this results in considerable variation in the actual cell-cell spacing and amount of cell-cell contact, and cannot prescribe the local environment. A more specific approach for modulating cell signaling is to use molecular inhibitors or genetic approaches to knock down specific signaling proteins, but both of these methods are best suited to manipulating small numbers of molecules. Here, we demonstrate a new approach to modulating cell-cell signaling that modulates the local environment of a cluster of cells by placing different numbers of cells at desired locations on a substrate. This method provides a complementary way to control the local diffusible and juxtacrine signaling between cells. Our method makes use of the Bio Flip Chip (BFC), a microfabricated silicone chip containing hundreds-to-thousands of microwells, each sized to hold either a single cell or small numbers of cells. We load the chip with cells simply by pipetting them onto the array of wells and washing unloaded cells off the array. The chip is then flipped onto a substrate, whereby the cells fall out of the wells and onto the substrate, maintaining their patterning. After the cells have attached, the chip can be removed (or left on). This approach to cell patterning is unique in that it: 1) doesn't alter the chemistry of the substrate, thus allowing cells to proliferate and migrate; 2) allows patterning onto any substrate, including tissue-culture polystyrene, glass, matrigel, and even feeder cell layers; and 3) is compatible with traditional microcontact printing, allowing the creation of extracellular matrix islands with cells placed inside those islands. In this video, we demonstrate the patterning of mouse embryonic stem cells onto tissue-culture polystyrene using the BFC.

We demonstrate a simple method for placing cells at desired locations on a substrate. This method patterns cells by flipping a silicone chip containing microwells filled with cells onto the substrate. This method provides a new way to modulate diffusible and juxtacrine signaling between cells.

图案的使用生物倒装芯片的胚胎干细胞

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such ...

科研

生物学

Flow-pattern Guided Fabrication of High-density Barcode Antibody Microarray

Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this report, we renovate the antibody microarray technology by using a novel approach for manufacturing and by introducing new features. The fabrication of our high-density antibody microarray is accomplished through perpendicularly oriented flow-patterning of single stranded DNAs and subsequent conversion mediated by DNA-antibody conjugates. This protocol outlines the critical steps in flow-patterning DNA, producing and purifying DNA-antibody conjugates, and assessing the quality of the fabricated microarray. The uniformity and sensitivity are comparable with conventional microarrays, while our microarray fabrication does not require the assistance of an array printer and can be performed in most research laboratories. The other major advantage is that the size of our microarray units is 10 times smaller than that of printed arrays, offering the unique capability of analyzing functional proteins from single cells when interfacing with generic microchip designs. This barcode technology can be widely employed in biomarker detection, cell signaling studies, tissue engineering, and a variety of clinical applications.

This protocol outlines the fabrication of a large-scale, multiplexed two-dimensional DNA or antibody array, with potential applications in cell signaling studies and biomarker detection.

高密度条码抗体芯片的水流模式的指导下制作

Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this ...

化学

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

在光刻技术定义的聚对二甲苯-C细胞图案：二氧化硅<sub&gt; 2</sub&gt;基板

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

生物工程

Mapping the Emergent Spatial Organization of Mammalian Cells using Micropatterns and Quantitative Imaging

A fundamental goal in biology is to understand how patterns emerge during development. Several groups have shown that patterning can be achieved in vitro when stem cells are spatially confined onto micropatterns, thus setting up experimental models which offer unique opportunities to identify, in vitro, the fundamental principles of biological organisation.
Here we describe our own implementation of the methodology. We adapted a photo-patterning technique to reduce the need for specialized equipment to make it easier to establish the method in a standard cell biology laboratory. We also developed a free, open-source and easy to install image analysis framework in order to precisely measure the preferential positioning of sub-populations of cells within colonies of standard shapes and sizes. This method makes it possible to reveal the existence of patterning events even in seemingly disorganized populations of cells. The technique provides quantitative insights and can be used to decouple influences of the environment (e.g., physical cues or endogenous signaling), on a given patterning process.

The method presented here uses micropatterning together with quantitative imaging to reveal spatial organization within mammalian cultures. The technique is easy to establish in a standard cell biology laboratory and offers a tractable system to study patterning in vitro.

使用微模式和定量成像绘制哺乳动物细胞的新兴空间组织图

A fundamental goal in biology is to understand how patterns emerge during development. Several groups have shown that patterning can be achieved in vitro ...

发育生物学

Patterning of Microorganisms and Microparticles through Sequential Capillarity-assisted Assembly

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, including studies of microbial physiology and interactions. At the simplest level, accurate spatial patterning of microorganisms would enable reliable, long-term imaging of large numbers of individual cells and transform the ability to quantitatively study distance-dependent microbe-microbe interactions. More uniquely, coupling accurate spatial patterning and full control over environmental conditions, as offered by microfluidic technology, would provide a powerful and versatile platform for single-cell studies in microbial ecology. 
This paper presents a microfluidic platform to produce versatile and user-defined patterns of microorganisms within a microfluidic channel, allowing complete optical access for long-term, high-throughput monitoring. This new microfluidic technology is based on capillarity-assisted particle assembly and exploits the capillary forces arising from the controlled motion of an evaporating suspension inside a microfluidic channel to deposit individual microsized objects in an array of traps microfabricated onto a polydimethylsiloxane (PDMS) substrate. Sequential depositions generate the desired spatial layout of single or multiple types of micro-sized objects, dictated solely by the geometry of the traps and the filling sequence. 
The platform has been calibrated using colloidal particles of different dimensions and materials: it has proven to be a powerful tool to generate diverse colloidal patterns and perform surface functionalization of trapped particles. Furthermore, the platform was tested on microbial cells, using Escherichia coli cells as a model bacterium. Thousands of individual cells were patterned on the surface, and their growth was monitored over time. In this platform, the coupling of single-cell deposition and microfluidic technology allows both geometric patterning of microorganisms and precise control of environmental conditions. It thus opens a window into the physiology of single microbes and the ecology of microbe-microbe interactions, as shown by preliminary experiments.

We present a technology that uses capillarity-assisted assembly in a microfluidic platform to pattern micro-sized objects suspended in a liquid, such as bacteria and colloids, into prescribed arrays on a polydimethylsiloxane substrate.

通过顺序毛细管辅助组装对微生物和微粒进行图案化

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, ...

工程学

A Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

图案蛋白质和细胞的通用的方法

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...

A Paired Bead and Magnet Array for Molding Microwells with Variable Concave Geometries

A spheroid culture is a useful tool for understanding cellular behavior in that it provides an in vivo-like three-dimensional environment. Various spheroid production methods such as non-adhesive surfaces, spinner flasks, hanging drops, and microwells have been used in studies of cell-to-cell interaction, immune-activation, drug screening, stem cell differentiation, and organoid generation. Among these methods, microwells with a three-dimensional concave geometry have gained the attention of scientists and engineers, given their advantages of uniform-sized spheroid generation and the ease with which the responses of individual spheroids can be monitored. Even though cost-effective methods such as the use of flexible membranes and ice lithography have been proposed, these techniques incur serious drawbacks such as difficulty in controlling the pattern sizes, achievement of high aspect ratios, and production of larger areas of microwells. To overcome these problems, we propose a robust method for fabricating concave microwells without the need for complex high-cost facilities. This method utilizes a 30 x 30 through-hole array, several hundred micrometer-order steel beads, and magnetic force to fabricate 900 microwells in a 3 cm x 3 cm polydimethylsiloxane (PDMS) substrate. To demonstrate the applicability of our method to cell biological applications, we cultured adipose stem cells for 3 days and successfully produced spheroids using our microwell platform. In addition, we performed a magnetostatic simulation to investigate the mechanism, whereby magnetic force was used to trap the steel beads in the through-holes. We believe that the proposed microwell fabrication method could be applied to many spheroid-based cellular studies such as drug screening, tissue regeneration, stem cell differentiation, and cancer metastasis.

This manuscript introduces a robust method of fabricating concave microwells without the need for complex high-cost facilities. Using magnetic force, steel beads, and a through-hole array, several hundred microwells were formed in a 3 cm x 3 cm polydimethylsiloxane (PDMS) substrate.

变凹形形状 Microwells 的配对磁珠和磁体阵列

A spheroid culture is a useful tool for understanding cellular behavior in that it provides an in vivo-like three-dimensional environment. Various ...

Fabrication of Magnetic Platforms for Micron-Scale Organization of Interconnected Neurons

The ability to direct neurons into organized neural networks has great implications for regenerative medicine, tissue engineering, and bio-interfacing. Many studies have aimed at directing neurons using chemical and topographical cues. However, reports of organizational control on a micron-scale over large areas are scarce. Here, an effective method has been described for placing neurons in preset sites and guiding neuronal outgrowth with micron-scale resolution, using magnetic platforms embedded with micro-patterned, magnetic elements. It has been demonstrated that loading neurons with magnetic nanoparticles (MNPs) converts them into sensitive magnetic units that can be influenced by magnetic gradients. Following this approach, a unique magnetic platform has been fabricated on which PC12 cells, a common neuron-like model, were plated and loaded with superparamagnetic nanoparticles. Thin films of ferromagnetic (FM) multilayers with stable perpendicular magnetization were deposited to provide effective attraction forces toward the magnetic patterns. These MNP-loaded PC12 cells, plated and differentiated atop the magnetic platforms, were preferentially attached to the magnetic patterns, and the neurite outgrowth was well aligned with the pattern shape, forming oriented networks. Quantitative characterization methods of the magnetic properties, cellular MNP uptake, cell viability, and statistical analysis of the results are presented. This approach enables the control of neural network formation and improves neuron-to-electrode interface through the manipulation of magnetic forces, which can be an effective tool for in vitro studies of networks and may offer novel therapeutic biointerfacing directions.

This work presents a bottom-up approach to the engineering of local magnetic forces for control of neuronal organization. Neuron-like cells loaded with magnetic nanoparticles (MNPs) are plated atop and controlled by a micro-patterned platform with perpendicular magnetization. Also described are magnetic characterization, MNP cellular uptake, cell viability, and statistical analysis.

为互连神经元微缩组织制造磁平台

The ability to direct neurons into organized neural networks has great implications for regenerative medicine, tissue engineering, and bio-interfacing. ...

Pattern Generation for Micropattern Traction Microscopy

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer length scale allows the use of these patterned contact zones for the measurement of traction forces, as each micropatterned dot allows for the formation of a single focal adhesion that then deforms the soft, underlying hydrogel. This approach has been used for a wide range of cell types, including endothelial cells, smooth muscle cells, fibroblasts, platelets, and epithelial cells. 
This review describes the evolution of techniques that allow the printing of extracellular matrix proteins onto polyacrylamide hydrogels in a regular array of dots of prespecified size and spacing. As micrometer-scale patterns are difficult to directly print onto soft substrates, patterns are first generated on rigid glass coverslips that are then used to transfer the pattern to the hydrogel during gelation. First, the original microcontact printing approach to generate arrays of small dots on the coverslip is described. A second step that removes most of the pattern to leave islands of small dots is required to control the shapes of cells and cell clusters on such arrays of patterned dots. 
Next, an evolution of this approach that allows for the generation of islands of dots using a single subtractive patterning step is described. This approach is greatly simplified for the user but has the disadvantage of a decreased lifetime for the master mold needed to make the patterns. Finally, the computational approaches that have been developed for the analysis of images of displaced dots and subsequent cell-generated traction fields are described, and updated versions of these analysis packages are provided.

We describe improvements to a standard method for measuring cellular traction forces, based on microcontact printing with a single subtractive patterning step of dot arrays of extracellular matrix proteins on soft hydrogels. This method allows for simpler and more consistent fabrication of island patterns, essential for controlling cell cluster shape.

微图案牵引显微镜的图案生成

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer ...

Stimulation of Patterned Neuronal Cultures by a Magnetic Field

Source: Stern, S., et al., <a href="https://app.jove.com/v/54357">External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures.</a> J. Vis. Exp. (2017)
This video demonstrates a method for stimulating neuronal cultures grown in a patterned circular ring on a coverslip, using a time-varying magnetic field.

磁场刺激模式化神经元培养物

1. Preparation of coverslips for 1D cultures.

	Clean glass coverslips by immersion in a base piranha solution consisting of 75 mL DDW, 25 mL 25% ammonia ...