这项研究由科学、ICT 和未来规划部 (NRF-2014R1A1A2057527 和 NRF-2016R1D1A1B03934418) 资助的韩国国家研究基金会 (NRF) 支持。

1. 通过孔阵列铝板和磁铁阵列的制备
<ol><li>准备两个50毫米 x 50 毫米 (或更大) 的铝板。每个板材的厚度为300µm, 是珠粒直径的一半。</li>
	<li>在其中一个铝板上形成一个 30 x 30 通孔阵列, 使用带有Φ550µm 微钻头的 CNC 旋转雕刻机, 其转速为30毫米/秒, 主轴速度为 8000 RPM。每个孔之间的距离 (中心到中心) 是 1 mm (图 1a和图 2a, i)。</li>
	<li>使用与 1.2 (图 1a和图 2a, ii) 中描述的程序相同的步骤, 在其他铝板上形成一个 30 x 30 阵列的Φ750-µm 通孔。</li>
	<li>在两个铝板的四角上, 使用粘胶带和形成Φ3 mm 对齐孔, 以彼此连接这两个板块。</li>
	<li>在15% 硫酸中浸泡12小时的铝板以清洁其表面。由于铝表面的薄层氧化铝使其具有耐腐蚀性, 因此该酸处理不会改变板材的孔径和厚度。</li>
	<li>形成一个 30 x 30 阵列的1个 1 x 1 毫米钕磁铁 (具有磁性强度为 0.363 N)。确保每个磁铁是相反的极性对它的邻居。为了防止磁体阵列的破裂或散射, 使用双面胶带 (图 2a、 iii 和嵌入图 2) 将 30 x 30 mm 的铝板连接到磁体阵列的底部。</li>
</ol>2. 磁珠捕集工艺
<ol><li>对齐和堆叠两个铝板 (顶板: 750-µm 孔板, 底板: 550-µm 孔板) 使用准备的对准孔在每个板块的四角 (图 1b)。</li>
	<li>通过将 M3 螺栓插入对准孔中, 将两个板锁定在一起, 然后用螺母固定螺栓 (图 1b)。</li>
	<li>堆叠在准备好的磁铁阵列上的铝板组件 (图 1b, 2b和2c)。在堆叠过程中, 将磁体阵列与铝板上的孔阵列对齐。然后使用粘胶带固定磁铁阵列的位置。</li>
	<li>在板组件上放置足够数量的φ mm SUJ2 钢珠, 并使用丙烯 (或非金属) 板对珠子进行操作, 使珠子在每个孔 (图 1c、 1d和1e) 同时被困住去除未在孔中的多余的珠子。</li>
	<li>小心取下顶部板, 以避免不必要的散射和错位的珠子 (图 1f)。</li>
</ol>3. 凹微制造
<ol><li>移动凹微模具, 生产步骤2.1 至 2.5, 以上, 以培养皿。</li>
	<li>混合烷 (硅橡胶) 单体和固化剂根据制造商的指示19与一个硅氧烷单体: 固化剂比10:1。</li>
	<li>通过使用干燥和真空泵来除去在该混合物中被困住的气泡, 以消除气体的混合物。</li>
	<li>使用与 3.3 (图 1f) 中描述的过程相同的步骤, 将该混合物倒入凹微模和脱气中。</li>
	<li>在80° c 的板上烘烤 2 h 的硅氧烷混合物, 形成一个珠嵌入的硅橡胶衬底 (图 1g)。</li>
	<li>从模具 (图 1g) 中卸下固化的硅基板。在去除过程中, 用洗涤瓶喷涂甲醇从模具中分离出硅烷基板。</li>
	<li>使用 15 mm x 2 mm 磁铁, 从硅基板上取下被夹住的钢珠 (图 1h)。在这个过程中, 任何足够强的磁铁从硅氧烷基板中提取珠子都可以使用。</li>
</ol>4. 球体培养
<ol><li>在本研究中, 用 24-井板上的Φ14 mm 活检穿孔, 切割凹微图案的硅橡胶衬底。</li>
	<li>在121° c 和 15 psi 的高压灭菌器中消毒所产生的Φ14 mm 的硅基板。</li>
	<li>将灭菌后的硅基板置于24井盘中。</li>
	<li>用 4% (w/v) pluronic F-127 溶液将整个硅烷基板涂上一整夜, 防止细胞附着在微表面。在涂层过程中, 通过移或使用超声波清洗器, 去除凹 microwells 中的气泡。</li>
	<li>使用磷酸盐缓冲盐水 (PBS) 冲洗 F-127 溶液三次。</li>
	<li>种子1毫升的细胞介质 (Dulbecco 氏改良的鹰培养基) 解决方案 (其中包含 2 x 106细胞) 在硅烷基板上。请注意, 播种密度可以根据目标球体的大小和/或目标细胞类型而改变。在这里, 脂肪来源的干细胞 (ASC) 被使用。</li>
	<li>用1000µL 吸管抽吸1毫升培养基, 以去除未被困在 microwells 中的多余细胞 (图 3)。</li>
	<li>孵育的细胞在36.5 ° c, 湿度的 > 95%, 和 5% CO2条件。在我们的研究中使用的陶瓷的情况下, 细胞聚集到一个球体在48小时。</li>
</ol>

<ol>
	<li>Fennema, E., Rivron, N., Rouwkema, J., van Blitterswijk, C., de Boer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spheroid+culture+as+a+tool+for+creating+3D+complex+tissues.">Spheroid culture as a tool for creating 3D complex tissues.</a> Trends Biotechnol. 31 (2), 108-115 (2013).</li><li>Djordjevic, B., Lange, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+spheroids+as+a+tool+for+prediction+of+radiosensitivity+in+tumor+therapy.">Hybrid spheroids as a tool for prediction of radiosensitivity in tumor therapy.</a> Indian J Exp Biol. 42 (5), 443-447 (2004).</li><li>Takezawa, T., Yamazaki, M., Mori, Y., Yonaha, T., Yoshizato, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+and+immuno-cytochemical+characterization+of+a+hetero-spheroid+composed+of+fibroblasts+and+hepatocytes.">Morphological and immuno-cytochemical characterization of a hetero-spheroid composed of fibroblasts and hepatocytes.</a> J Cell Sci. 101 (3), 495-501 (1992).</li><li>Gottfried, E., Kunz-Schughart, L. A., Andreesen, R., Kreutz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brave+little+world:+spheroids+as+an+in+vitro+model+to+study+tumor-immune-cell+interactions.">Brave little world: spheroids as an in vitro model to study tumor-immune-cell interactions.</a> Cell Cycle. 5 (7), 691-695 (2006).</li><li>Zhang, X., 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=Development+of+an+in+vitro+multicellular+tumor+spheroid+model+using+microencapsulation+and+its+application+in+anticancer+drug+screening+and+testing.">Development of an in vitro multicellular tumor spheroid model using microencapsulation and its application in anticancer drug screening and testing.</a> Biotechnol Prog. 21 (4), 1289-1296 (2005).</li><li>Kim, B. C., 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=Microwell-mediated+micro+cartilage-like+tissue+formation+of+adipose-derived+stem+cell.">Microwell-mediated micro cartilage-like tissue formation of adipose-derived stem cell.</a> Macromol Res. 22 (3), 287-296 (2014).</li><li>Fatehullah, A., Tan, S. H., Barker, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoids+as+an+in+vitro+model+of+human+development+and+disease.">Organoids as an in vitro model of human development and disease.</a> Nature cell biology. 18 (3), 246-254 (2016).</li><li>Yuhas, J. M., Li, A. P., Martinez, A. O., Ladman, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+method+for+production+and+growth+of+multicellular+tumor+spheroids.">A simplified method for production and growth of multicellular tumor spheroids.</a> Cancer Res. 37 (10), 3639-3643 (1977).</li><li>Hamilton, G. A., Westmoreland, C., George, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+medium+composition+on+the+morphology+and+function+of+rat+hepatocytes+cultured+as+spheroids+and+monolayers.">Effects of medium composition on the morphology and function of rat hepatocytes cultured as spheroids and monolayers.</a> In Vitro Cell Dev Biol-Animal. 37 (10), 656-667 (2001).</li><li>Nyberg, S. L., 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,+large-scale+formation+of+porcine+hepatocyte+spheroids+in+a+novel+spheroid+reservoir+bioartificial+liver.">Rapid, large-scale formation of porcine hepatocyte spheroids in a novel spheroid reservoir bioartificial liver.</a> Liver Transplant. 11 (8), 901-910 (2005).</li><li>Lazar, 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=Extended+liver-specific+functions+of+porcine+hepatocyte+spheroids+entrapped+in+collagen+gel.">Extended liver-specific functions of porcine hepatocyte spheroids entrapped in collagen gel.</a> In Vitro Cell Dev Biol-Animal. 31 (5), 340-346 (1995).</li><li>Kelm, J. M., Timmins, N. E., Brown, C. J., Fussenegger, M., Nielsen, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+generation+of+homogeneous+multicellular+tumor+spheroids+applicable+to+a+wide+variety+of+cell+types.">Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types.</a> Biotechnol Bioeng. 83 (2), 173-180 (2003).</li><li>Lin, R. Z., Chang, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+three-dimensional+multicellular+spheroid+culture+for+biomedical+research.">Recent advances in three-dimensional multicellular spheroid culture for biomedical research.</a> Biotechnol J. 3 (9-10), 1172-1184 (2008).</li><li>Choi, Y. Y., 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=Controlled-size+embryoid+body+formation+in+concave+microwell+arrays.">Controlled-size embryoid body formation in concave microwell arrays.</a> Biomaterials. 31 (15), 4296-4303 (2010).</li><li>Hwang, J. W., 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=Functional+clustering+of+pancreatic+islet+cells+using+concave+microwell+array.">Functional clustering of pancreatic islet cells using concave microwell array.</a> Macromol Res. 19 (12), 1320-1326 (2011).</li><li>Wong, S. F., 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=Concave+microwell+based+size-controllable+hepatosphere+as+a+three-dimensional+liver+tissue+model.">Concave microwell based size-controllable hepatosphere as a three-dimensional liver tissue model.</a> Biomaterials. 32 (32), 8087-8096 (2011).</li><li>Yeon, S. E., 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=Application+of+concave+microwells+to+pancreatic+tumor+spheroids+enabling+anticancer+drug+evaluation+in+a+clinically+relevant+drug+resistance+model.">Application of concave microwells to pancreatic tumor spheroids enabling anticancer drug evaluation in a clinically relevant drug resistance model.</a> PloS one. 8 (9), (2013).</li><li>Park, J. Y., Hwang, C. M., Lee, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ice-lithographic+fabrication+of+concave+microwells+and+a+microfluidic+network.">Ice-lithographic fabrication of concave microwells and a microfluidic network.</a> Biomed Microdevices. 11 (1), 129-133 (2009).</li><li>Corning, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Sylgard 184 Silicone Elastomer. Technical Data Sheet. , (2008).</li><li>Giang, U. B. T., Lee, D., King, M. R., DeLouise, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfabrication+of+cavities+in+polydimethylsiloxane+using+DRIE+silicon+molds.">Microfabrication of cavities in polydimethylsiloxane using DRIE silicon molds.</a> Lab on a Chip. 7 (12), 1660-1662 (2007).</li><li>Choi, J. 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=Capture+and+culturing+of+single+microalgae+cells,+and+retrieval+of+colonies+using+a+perforated+hemispherical+microwell+structure.">Capture and culturing of single microalgae cells, and retrieval of colonies using a perforated hemispherical microwell structure.</a> RSC Advances. 4 (106), 61298-61304 (2014).</li><li>Zhong, K., Gao, Y., Li, F., Zhang, Z., Luo, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+PDMS+microlens+array+by+digital+maskless+grayscale+lithography+and+replica+molding+technique.">Fabrication of PDMS microlens array by digital maskless grayscale lithography and replica molding technique.</a> Optik. 125 (10), 2413-2416 (2014).</li><li>Lai, D., 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=Simple+multi-level+microchannel+fabrication+by+pseudo-grayscale+backside+diffused+light+lithography.">Simple multi-level microchannel fabrication by pseudo-grayscale backside diffused light lithography.</a> RSC advances. 3 (42), 19467-19473 (2013).</li><li>Pan, J., 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=Fabrication+of+a+3D+hair+follicle-like+hydrogel+by+soft+lithography.">Fabrication of a 3D hair follicle-like hydrogel by soft lithography.</a> J Biomed MAter Res A. 101 (11), 3159-3169 (2013).</li><li>Mori, R., Sakai, Y., Nakazawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropatterned+organoid+culture+of+rat+hepatocytes+and+HepG2+cells.">Micropatterned organoid culture of rat hepatocytes and HepG2 cells.</a> J Biosci Bioeng. 106 (3), 237-242 (2008).</li></ol>

作者没有利益冲突透露。

这种制作方法面临的主要挑战是在铝板的通孔阵列中的珠子的安全固定。为了解决这一难题, 以 30 x 30 磁铁阵列的形式使用磁力来稳固地固定珠子, 如图 6和7所示。磁体阵列的磁通密度与极性相反, 在每个磁体表面的中心最强。因为磁力的强度取决于磁通密度, 所以珠子被引导到每个磁铁的顶面中心, 在那里他们被放置在位置。如果使用一个大尺寸的磁铁 (5 厘米 x 5 cm x 1 厘?...

生长成球状形态的细胞比二维平面区域性1更类似于实体组织。考虑到这一优势, 采用椭球的方法来改善细胞与细胞相互作用的研究2,3, 免疫激活4, 药物筛选5, 和差异性6。此外, 椭球结合多种细胞类型最近已应用于 organoids (近生理三维 (3D) 组织), 这是非常有用的研究人类发展和疾病7。几种方法已被用于生产椭球。最简单的方法是利用非粘合表面, 使细胞聚集在一起, 形成椭球。培养皿可以用牛血清白蛋白、pluronic F-127 或疏水性聚合物 (如聚 2-乙甲基丙烯酸酯) 来处理, 使其表面不粘附8, 9。微调烧瓶方法是另一种众所周知的生产大量椭球10,11的方式。在这种方法中, 细胞通过搅拌来保持悬浮, 以防止它们附着在基体上。相反, 浮动单元格聚合以形成椭球。无粘结面法和微调瓶法均能产生大量的椭球。但是, 它们受到限制, 包括控制球体尺寸的困难, 以及每个球体的跟踪和监测。作为对此类问题的一种补救方法, 另一种球形生产方式, 即悬挂跌落法可以采用12。这包括将细胞悬浮液沉淀在培养皿盖子的下侧。这些下落通常是15到30µL 在大小并且包含大约300到3000个细胞13。当盖子是倒置的, 下落由表面张力举行。每个下落的微重力环境集中细胞, 然后形成唯一椭球在自由的液体空气接口。悬挂下降法的优点是它提供了一个受控的大小分布, 而它很容易跟踪和监测每个球体, 相对于非胶粘剂表面和微调烧瓶的方法。然而, 这种方法在椭球的大量生产和生产过程本身是过度劳动密集型的情况下产生了一个不利因素。
微阵列是一个平板, 有许多微尺寸的井, 每一个直径不等, 从100到1000µm。在使用 microwells 时, 球体的生产原理与非粘性表面方法相似。好处包括 microwells 提供空间之间的 microwells 分离的细胞或椭球, 这样, 它是易于控制的球体大小, 同时也使它易于监测每个单一的球体。随着大量的 microwells, 高吞吐量球体的生产也成为可能。microwells 的另一个优势是根据用户独特的实验目的, 选择不同形状的水井 (六面体、圆柱形、三角形棱柱)。然而, 一般来说, 一个三维 (3D) 凹 (或半球) 形状被认为是最适合生产统一尺寸的单椭球。因此, 凹 microwells 的用处已经报告了许多细胞生物学研究, 如那些检测胚胎干细胞分化的心肌细胞14, 胰岛细胞簇的胰岛素分泌,15,肝细胞酶活性为16, 肿瘤耐药性椭球17。
不幸的是, 制作 microwells 往往需要专门的微设施;传统的光刻方法需要曝光和开发设备, 而反应离子蚀刻的方法需要等离子和离子束设备。这类设备费用高昂, 加上复杂的制作过程, 给无法进入微的生物学家带来了高门槛。为了克服这些问题, 提出了其他成本效益高的方法, 如冰光刻18 (使用冷冻水滴) 和柔性膜法14 (使用膜、通孔衬底和真空)。然而, 这些方法也有严重的缺点, 如难以控制的模式大小, 实现高宽比, 和生产大面积 microwells。
为了克服上述问题, 我们提出了一种新的凹微制造方法, 利用孔基板、钢珠和磁铁阵列。利用这种方法, 利用磁力辅助的自锁金属珠 (图 1) 的机理, 可以制作数以百计的凹球形 microwells。制作过程涉及使用极少数昂贵和复杂的设施, 不需要许多先进的技能。因此, 即使是不熟练的人也很容易承担这种制作方法。为了证明所提出的方法, 在凹 microwells 培养人脂肪干细胞, 产生椭球。

<table><tbody><tr><td>CNC rotary engraver</td><td>Roland DGA</td><td>EGX-350</td><td></td></tr><tr><td>Micro drill bit</td><td>HAM Pr&amp;auml;zision</td><td>30-1301 TA</td><td>&amp;Phi; 0.55 and 0.75 mm</td></tr><tr><td>Sulfuric acid 98%</td><td>Daejung</td><td>7683-4100</td><td>For cleaning aluminum plate.&lt;br/&gt; Dilute with distilled water with 15% solution</td></tr><tr><td>Neodymium magnet</td><td>Supermagnete</td><td>W-01-N</td><td>1 x 1 x 1 mm</td></tr><tr><td>Bearing ball</td><td>Agami Modeling</td><td>SUJ2</td><td>&amp;Phi; 600 &amp;mu;m steel bead</td></tr><tr><td>Polydimethylsiloxane (PDMS)</td><td>Dowcorning</td><td>Sylgard 184</td><td></td></tr><tr><td>Pluronic F-127</td><td>Sigma Aldrich</td><td>p2443</td><td>Dilute with phosphate buffered saline to 4% (w/v) solution</td></tr><tr><td>Dulbecco&#039;s modified eagle&#039;s medium (DMEM)</td><td>ATCC</td><td>30-2002</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate buffered saline (D-PBS)</td><td>ATCC</td><td>30-2200</td><td></td></tr><tr><td>Fetal bovine serum</td><td>ATCC</td><td>30-2020</td><td></td></tr><tr><td>Adipose-derived mesenchymal stem cells</td><td>ATCC</td><td>ATCC PCS-500-011</td><td></td></tr></tbody></table>

a paired bead and magnet array for molding microwells with variable concave geometries

椭球区域性是理解细胞行为的有用工具, 因为它提供了一个在体内类似的三维环境。各种球体的生产方法, 如无粘结表面, 微调烧瓶, 悬挂滴, 和 microwells 已被用于研究细胞间的相互作用, 免疫活化, 药物筛选, 干细胞分化, 和化生成。在这些方法中, microwells 与三维凹几何已经得到了科学家和工程师的注意, 由于他们的优势, 统一大小的球体生成和轻松的反应, 个别椭球可以监测.尽管提出了使用柔性膜和冰光刻等成本效益高的方法, 但这些技术存在着严重的缺点, 如难以控制模式尺寸、实现较高的长宽比和生产大面积的 microwells。针对这些问题, 提出了一种在不需要复杂的高成本设备的情况下制作凹 microwells 的鲁棒方法。该方法利用了 30 x 30 通孔阵列、几百微米级的钢珠和磁力, 在 3 cm x 3 cm 烷 (microwells) 基底上制造了900个。为了证明我们的方法对细胞生物学应用的适用性, 我们培养脂肪干细胞3天, 成功地生产椭球使用我们的微平台。此外, 我们进行了一个静磁场模拟研究的机制, 即磁力被用来捕获钢珠在通孔。我们认为, 所提出的微制备方法可以应用于许多基于球体的细胞研究, 如药物筛选、组织再生、干细胞分化和肿瘤转移。

通过以下步骤2.1 到 3.7, 成功地制作了凸模和微模式。(图 4)。商业钢珠被困在 30 x 30 通孔阵列。珠子被紧紧地抱着, 珠子和相应的通孔之间没有缝隙 (图 4a)。预制凹微的形状为凹半球形, 直径为600µm, 与钢珠相同 (图 4b)。凹微的横截面 (图 4c) 显示相邻微的距离为1毫米 (中心至中心), 与?...

Watch this Scientific Journal Video about A Paired Bead and Magnet Array for Molding Microwells with Variable Concave Geometries at JoVE.com

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

本文介绍了一种在不需要复杂的高成本设备的情况下, 制作凹 microwells 的鲁棒方法。采用磁力、钢珠和通孔阵列, 在 3 cm x 3 cm 烷 (microwells) 衬底上形成几百个。

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 ...

a-paired-bead-magnet-array-for-molding-microwells-with-variable

Universidad de Cartagena UDEC

Research

Education

JoVE Core

JoVE Journal

Physics

Bioengineering

Unit 1

Magnetic Forces and Fields

SCIENTISTS IN ACTION

8.6K 次观看.  Chung-Ang University. 本文介绍了一种在不需要复杂的高成本设备的情况下, 制作凹 microwells 的鲁棒方法。采用磁力、钢珠和通孔阵列, 在 3 cm x 3 cm 烷 (microwells) 衬底上形成几百个。

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

Microbubble Fabrication of Concave-porosity PDMS Beads

Microbubble fabrication (by use of a fine emulsion) provides a means of increasing the surface-area-to-volume (SAV) ratio of polymer materials, which is particularly useful for separations applications. Porous polydimethylsiloxane (PDMS) beads can be produced by heat-curing such an emulsion, allowing the interface between the aqueous and aliphatic phases to mold the morphology of the polymer. In the procedures described here, both polymer and crosslinker (triethoxysilane) are sonicated together in a cold-bath sonicator. Following a period of cross-linking, emulsions are added dropwise to a hot surfactant solution, allowing the aqueous phase of the emulsion to separate, and forming porous polymer beads. We demonstrate that this method can be tuned, and the SAV ratio optimized, by adjusting the electrolyte content of the aqueous phase in the emulsion. Beads produced in this way are imaged with scanning electron microscopy, and representative SAV ratios are determined using Brunauer&#8211;Emmett&#8211;Teller (BET) analysis. Considerable variability with the electrolyte identity is observed, but the general trend is consistent: there is a maximum in SAV obtained at a specific concentration, after which porosity decreases markedly.

Procedures used to generate microstructured concave-porosity polydimethylsiloxane beads are presented. Effects of electrolyte concentration and identity within the aqueous phase are particularly emphasized.

的凹孔隙PDMS珠微泡制作

Microbubble fabrication (by use of a fine emulsion) provides a means of increasing the surface-area-to-volume (SAV) ratio of polymer materials, which is ...

科研

化学

Shrinky-Dink Hanging Drops: A Simple Way to Form and Culture Embryoid Bodies

Embryoid bodies (EB) are aggregates of embryonic stem cells. The most common way of creating these aggregates is the hanging drop method, a laborious approach of pipetting an arbitrary number of cells into well plates. The interactions between the stem cells forced into close proximity of one another promotes the generation of the EBs. Because the media in each of the wells has to be manually exchanged every day, this approach is manually intensive.
Moreover, because environmental parameters including cell-cell, cell-soluble factor interactions, pH, and oxygen availability can be functions of EB size, cell populations obtained from traditional hanging drops can vary dramatically even when cultured under identical conditions. Recent studies have indeed shown that the initial number of cells forming the aggregate can have significant effects on stem cell differentiation. We have developed a simple, rapid, and scalable culture method to load pre-defined numbers of cells into microfabricated wells and maintain them for embryoid body development. Finally, these cells are easily accessible for further analysis and experimentation. This method is amenable to any lab and requires no dedicated equipment. We demonstrate this method by creating embryoid bodies using a red fluorescent mouse cell line (129S6B6-F1).

We show a simple and rapid method to load pre-defined numbers of cells into microfabricated wells and maintain them for embryoid body development.

Shrinky丁克挂滴：一个简单的方法，形式和文化胚体

Embryoid bodies (EB) are aggregates of embryonic stem cells. The most common way of creating these aggregates is the hanging drop method, a laborious ...

生物学

Creating Sub-50 Nm Nanofluidic Junctions in PDMS Microfluidic Chip via Self-Assembly Process of Colloidal Particles

Polydimethylsiloxane (PDMS) is the prevailing building material to make microfluidic devices due to its ease of molding and bonding as well as its transparency. Due to the softness of the PDMS material, however, it is challenging to use PDMS for building nanochannels. The channels tend to collapse easily during plasma bonding. In this paper, we present an evaporation-driven self-assembly method of silica colloidal nanoparticles to create nanofluidic junctions with sub-50 nm pores between two microchannels. The pore size as well as the surface charge of the nanofluidic junction is tunable simply by changing the colloidal silica bead size and surface functionalization outside of the assembled microfluidic device in a vial before the self-assembly process. Using the self-assembly of nanoparticles with a bead size of 300 nm, 500 nm, and 900 nm, it was possible to fabricate a porous membrane with a pore size of ~45 nm, ~75 nm and ~135 nm, respectively. Under electrical potential, this nanoporous membrane initiated ion concentration polarization (ICP) acting as a cation-selective membrane to concentrate DNA by ~1,700 times within 15 min. This non-lithographic nanofabrication process opens up a new opportunity to build a tunable nanofluidic junction for the study of nanoscale transport processes of ions and molecules inside a PDMS microfluidic chip.

We propose a simple self-assembly technique of silica colloidal nanoparticles to create a nanofluidic junction between two microchannels in polydimethylsiloxane (PDMS). Using this technique, a nanoporous bead membrane with a pore size down to ~45 nm was built inside a microchannel and applied to electrokinetic preconcentration of DNA samples.

通过胶体粒子自组装过程创建在PDMS微流控芯片分50纳米的纳米流体结

Polydimethylsiloxane (PDMS) is the prevailing building material to make microfluidic devices due to its ease of molding and bonding as well as its ...

工程学

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are heterogeneous mixtures of many types of lipids, contain membrane-bound proteins and are decorated with carbohydrates. In some experiments, it is desirable to decouple the biophysical or biochemical properties of the lipid bilayer from those of the natural membrane. Such cases call for the use of model systems such as giant vesicles, liposomes or supported lipid bilayers (SLBs). Arrays of SLBs are particularly attractive for sensing applications and mimicking cell-cell interactions. Here we describe a new method for forming SLB arrays. Submicron-diameter SiO2 beads are first coated with lipid bilayers to form spherical SLBs (SSLBs). The beads are then deposited into an array of micro-fabricated submicron-diameter microwells. The preparation technique uses a "squeegee" to clean the substrate surface, while leaving behind SSLBs that have settled into microwells. This method requires no chemical modification of the microwell substrate, nor any particular targeting ligands on the SSLB. Microwells are occupied by single beads because the well diameter is tuned to be just larger than the bead diameter. Typically, more 75% of the wells are occupied, while the rest remain empty. In buffer SSLB arrays display long-term stability of greater than one week. Multiple types of SSLBs can be placed in a single array by serial deposition, and the arrays can be used for sensing, which we demonstrate by characterizing the interaction of cholera toxin with ganglioside GM1. We also show that phospholipid vesicles without the bead supports and biomembranes from cellular sources can be arrayed with the same method and cell-specific membrane lipids can be identified.

Supported lipid bilayers and natural membrane particles are convenient systems that can approximate the properties of cell membranes and be incorporated in a variety of analytical strategies. Here we demonstrate a method for preparing microarrays composed of supported lipid bilayer-coated SiO2 beads, phospholipid vesicles or natural membrane particles.

生物膜的微阵列用刮墨刀型组合方式形成

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are ...

生物工程

Polymeric Microneedle Array Fabrication by Photolithography

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a photomask consisting of embedded micro-lenses. Embedded micro-lenses were found to influence MN geometry (sharpness). Robust MN arrays with tip diameters ranging between 41.5 &#181;m &#177; 8.4 &#181;m and 71.6 &#181;m &#177; 13.7 &#181;m, with two different lengths (1,336 &#181;m &#177; 193 &#181;m and 957 &#181;m &#177; 171 &#181;m) were fabricated. These MN arrays may provide potential applications in delivery of low molecular and macromolecular therapeutic agents through skin.

Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography.

聚合物微针阵列制造用光刻

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a ...

Easy and Accurate Mechano-profiling on Micropost Arrays

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate surroundings. Particularly, cell-substrate interactions are the main interest. Today micropost arrays are a well-characterized and established method with a broad range of applications that have been published over the last decade. However, there seems to be a reservation among biologists to adapt the technique due to the lengthy and challenging process of micropost manufacture along with the lack of easily approachable software for analyzing images of cells interacting with microposts. 
The force read-out from microposts is surprisingly easy. A micropost acts like a spring with the cell ideally attached at its tip. Depending on size a cell applies force from its cytoskeleton through one or multiple focal adhesion points to the micropost, thus deflecting the micropost. The amount of deflection correlates directly to the applied force in direction and in magnitude. The number of microposts covered by a cell and the post deflection patterns are characteristic and allow determination of values like force per post and many biologically relevant parameters that allow &#8220;mechano-profiling&#8221; of cell phenotypes.
A convenient method for mechano-profiling is described here combining the first generation of ready-to-use commercially available microposts with an in-house developed software package that is now accessible to all researchers. As a demonstration of typical application, single images of bone cancer cells were taken in bright-field microscopy for mechano-profiling of cell line models of metastasis. This combination of commercial traction force sensors and open source software for analysis allows for the first time a rapid implementation of the micropost array technique into routine lab work done by non-expert users. Furthermore, a robust and streamlined analysis process enables a user to analyze a large number of micropost images in a highly time-efficient manner.

Described are protocols for quantifying mechanical interactions between adherent cells and microstructured substrates. These interactions are closely linked to essential cell behaviors including migration, proliferation, differentiation, and apoptosis. The protocols present an open-source image analysis software called MechProfiler, which enables determination of involved forces for each micropost.

在Micropost阵列方便，准确机械力分析

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

缩微和3D微血管大会

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Computer Numerical Control Micromilling of a Microfluidic Acrylic Device with a Staggered Restriction for Magnetic Nanoparticle-Based Immunoassays

Microfluidic systems have greatly improved immunoassay techniques. However, many microfabrication techniques require specialized, expensive, or complicated equipment, making fabrication costly and incompatible with mass production, which is one of the most important preconditions for point-of-care tests (POCT) to be adopted in low-resource settings. This work describes the fabrication process of an acrylic (polymethylmethacrylate, PMMA) device for nanoparticle-conjugated enzymatic immunoassay testing using the computer numerical control (CNC) micromilling technique. The functioning of the microfluidic device is shown by performing an immunoassay to detect a commercial antibody using lysozyme as a model antigen conjugated to 100 nm magnetic nanoparticles. This device integrates a physical staggered restriction of only 5 &#181;m in height, used to capture magnetic microparticles that make up a magnetic trap by placing an external magnet. In this way, the magnetic force on the immunosupport of conjugated nanoparticles is enough to capture them and resist flow drag. This microfluidic device is particularly suitable for low-cost mass production without the loss of precision for immunoassay performance.

Microfluidics is a powerful tool for the development of diagnostic tests. However, expensive equipment and materials, as well as laborious fabrication and handling techniques, are often required. Here, we detail the fabrication protocol of an acrylic microfluidic device for magnetic micro- and nanoparticle-based immunoassays in a low-cost and simple-to-use setting.

基于磁性纳米颗粒免疫分析的交错限制微流控亚克力装置的计算机数控微铣削

Microfluidic systems have greatly improved immunoassay techniques. However, many microfabrication techniques require specialized, expensive, or ...

High-Speed Magnetic Tweezers for Nanomechanical Measurements on Force-Sensitive Elements

Single-molecule magnetic tweezers (MTs) have served as powerful tools to forcefully interrogate biomolecules, such as nucleic acids and proteins, and are therefore poised to be useful in the field of mechanobiology. Since the method commonly relies on image-based tracking of magnetic beads, the speed limit in recording and analyzing images, as well as the thermal fluctuations of the beads, has long hampered its application in observing small and fast structural changes in target molecules. This article describes detailed methods for the construction and operation of a high-resolution MT setup that can resolve nanoscale, millisecond dynamics of biomolecules and their complexes. As application examples, experiments with DNA hairpins and SNARE complexes (membrane-fusion machinery) are demonstrated, focusing on how their transient states and transitions can be detected in the presence of piconewton-scale forces. We expect that high-speed MTs will continue to enable high-precision nanomechanical measurements on molecules that sense, transmit, and generate forces in cells, and thereby deepen our molecular-level understanding of mechanobiology.

Here, we describe a high-speed magnetic tweezer setup that performs nanomechanical measurements on force-sensitive biomolecules at the maximum rate of 1.2 kHz. We introduce its application to DNA hairpins and SNARE complexes as model systems, but it will be also applicable to other molecules involved in mechanobiological events.

高速磁镊，用于力敏元件的纳米力学测量

Single-molecule magnetic tweezers (MTs) have served as powerful tools to forcefully interrogate biomolecules, such as nucleic acids and proteins, and are ...

生物化学

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

多路人工细胞微环境阵列的制备

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...