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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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. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stencil+micropatterning+of+human+pluripotent+stem+cells+for+probing+spatial+organization+of+differentiation+fates.">Stencil micropatterning of human pluripotent stem cells for probing spatial organization of differentiation fates.</a> J Vis Exp. 112, e54097 (2016).</li><li>Joddar, B., 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=Engineering+approaches+for+cardiac+organoid+formation+and+their+characterization.">Engineering approaches for cardiac organoid formation and their characterization.</a> Transl Res. 250, 46-67 (2022).</li></ol>

提交人没有相互竞争的经济利益。

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#215; 50 mm</td>
		</tr>
		<tr>
			<td>DiD</td>
			<td>MedChemExpress (MCE)&#160;</td>
			<td>HY-D1028</td>
			<td>For labeling cells with red fluorescence (Ex: 640 nm)</td>
		</tr>
		<tr>
			<td>DiI</td>
			<td>MedChemExpress (MCE)&#160;</td>
			<td>HY-D0083&#160;</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&#160;</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 &#215; 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

1.2K 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的...

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 

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

等离子体表面图案光刻技术的蜂窝网络创作

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

科研

生物工程

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

TIRF microscopy has emerged as a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules in vitro and in living cells1. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface2. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching2,3. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups.
At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures4-8. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells.
We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.

Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.

皮层组织和动态可视化的微生物，利用全内反射荧光显微镜

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

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, ...

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 ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

We demonstrate the use of patterned aerosol adhesives to construct 3D paper microfluidic devices. This method of adhesive application forms semi-permanent bonds between layers, enabling single-use devices to be non-destructively disassembled after use and to ease folding complex nonplanar structures.

使用粘性图案构建3D纸微流体装置

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one particular implementation of the MFP, the hierarchical hydrodynamic flow confinement (hHFC), multiple liquids are simultaneously brought in contact with a substrate. Local chemical action and liquid shaping using the hHFC, is exploited to create cell patterns by locally lysing and removing cells. By utilizing the scanning ability of the MFP, user-defined patterns of cell monolayers are created. This protocol enables rapid, real-time and spatially controlled cell patterning, which can allow selective cell-cell and cell-matrix interaction studies.

We present a protocol to perform subtractive patterning of live cell monolayers on a surface. This is achieved by local and selective lysis of adherent cells using a microfluidic probe (MFP). The cell lysate retrieved from local regions can be used for downstream analysis, enabling molecular profiling studies.

活细胞层的快速消减带图案微流控探头

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

用于细胞培养应用光介导的形成和构图的水凝胶

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

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 ...

Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors within the medium to control cell behavior. However, soluble additions cannot mimic certain signaling motifs, such as matrix-bound growth factors, cell-cell signaling, and spatial biochemical cues, which are common influences on cells. Furthermore, biophysical properties of the matrix, such as substrate stiffness, play important roles in cell fate, which is not easily manipulated using conventional cell culturing practices. In this method, we describe a straightforward protocol to provide patterned bioactive proteins on synthetic polyethylene glycol (PEG) hydrogels using photochemistry. This platform allows for the independent control of substrate stiffness and spatial biochemical cues. These hydrogels can achieve a large range of physiologically relevant stiffness values. Additionally, the surfaces of these hydrogels can be photopatterned with bioactive peptides or proteins via thiol-ene click chemistry reactions. These methods have been optimized to retain protein function after surface immobilization. This is a versatile protocol that can be applied to any protein or peptide of interest to create a variety of patterns. Finally, cells seeded onto the surfaces of these bioactive hydrogels can be monitored over time as they respond to spatially specific signals.

In this method, we use photopolymerization and click chemistry techniques to create protein or peptide patterns on the surface of polyethylene glycol (PEG) hydrogels, providing immobilized bioactive signals for the study of cellular responses in vitro.

生物活性蛋白或多肽在水凝胶上的应用

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors ...

High-resolution Patterning Using Two Modes of Electrohydrodynamic Jet: Drop on Demand and Near-field Electrospinning

Electrohydrodynamic (EHD) jet printing has drawn attention in various fields because it can be used as a high-resolution and low-cost direct patterning tool. EHD printing uses a fluidic supplier to maintain the extruded meniscus by pushing the ink out of the nozzle tip. The electric field is then used to pull the meniscus down to the substrate to produce high-resolution patterns. Two modes of EHD printing have been used for fine patterning: continuous near-field electrospinning (NFES) and dot-based drop-on-demand (DOD) EHD printing. According to the printing modes, the requirements for the printing equipment and ink viscosity will differ. Even though two different modes can be implemented with a single EHD printer, the realization methods significantly differ in terms of ink, fluidic system, and driving voltage. Consequently, without a proper understanding of the jetting requirements and limitations, it is difficult to obtain the desired results. The purpose of this paper is to present a guideline so that inexperienced researchers can reduce the trial and error efforts to use the EHD jet for their specific research and development purposes. To demonstrate the fine-patterning implementation, we use Ag nanoparticle ink for the conductive patterning in the protocol. In addition, we also present the generalized printing guidelines that can be used for other types of ink for various fine-patterning applications.

Here, we present a protocol to produce high-resolution conductive patterns using electrohydrodynamic (EHD) jet printing. The protocol includes two modes of EHD jet printing: the continuous near-field electrospinning (NFES) and the dot-based drop-on-demand (DOD) EHD printing.

采用两种 Electrohydrodynamic 射流的高分辨率模式: 需求下降和近场静电纺丝

Electrohydrodynamic (EHD) jet printing has drawn attention in various fields because it can be used as a high-resolution and low-cost direct patterning ...