这项工作是支持由库尔特基金会，Beyster基金会，ATA和美国国家科学基金会研究生研究奖学金（批准号DGE 0718128;编号:2010101926）JBW暑期班的本科生研究机会（UROP）。

1。相体系相分离的特性:确定阈值<ol><li>准备含有PEG和DEX的解决方案中所需的缓冲区或细胞培养基中所示，在图1（紫色的点）在15毫升或50毫升锥形管。在下文中，PEG和DEX将参考35 kDa的PEG和500 kDa的DEX;然而，临界浓度会改变，这取决于所用的两种聚合物。记录每个解决方案中的质量PEG和DEX。高浓度聚合物解决方案可能需要几个小时才能溶解。无血清的解决方案可用于涡旋。含有蛋白或血清，把试管放在两种聚合物的摇摆阶段，直到完全溶解。记录的重量，所使用的介质的初始浓度溶解的聚合物和注意到。 </li><li>一旦聚合物充分溶解，解决方案应该会出现混浊。这是第一个迹象表明已发生相分离。为了证实这一点，让聚合物溶液在一个垂直的位置，在室温下20分钟的休息。离心以1,000 xg离心可以用于加速的相分离过程。密集的底部阶段将DEX丰富，顶部阶段将PEG丰富。 </li><li>慢慢地添加额外的缓冲或媒体的管。应使用小的增量，以便不进行过冲的相分离点。 </li><li>当该溶液变得清晰和不再相离心后分离，相分离的阈值已被达到。记录管的最终重量。 </li><li>使用以前的记录的权重的聚合物，随着添加媒体后的最终重量，确定％wt / wt的每两个在该相分离的聚合物中不再出现。 </li><li>绘制这些值作为％wt / wt的PEG对y轴和％wt / wt的DEX在x-轴。称为双结点曲线，此图中，可以用来确定的阈值浓度的相分离为不同的concentrati在一个特定的细胞培养基中的PEG / DEX附加。 </li></ol> 2。配置1:排除图案（96孔板格式） <ol><li>准备单独的解决方案，为5.0％wt / wt的PEG和12.8％wt / wt的DEX细胞培养液中。使用双水相的至少两倍的临界点的解决方案，以确保保持ATPS后的聚合物相对于彼此平衡。有一个小的量的磁通的DEX到的富含PEG的相，反之亦然，因此，工作太附近的临界浓度，可导致损失的浓度下降到低于临界点的相位系统。同样地，将细胞培养皿中的湿度和温度控制培养箱可以改变两相性能，使两种溶液混溶的。注:某些细胞可能有更好的表现与其他ATPS配方。上可接受的制剂，可以选择是根据从第1部分上的双结点曲线确定。 </li><li>收获细胞，用于排斥patterning。确定可用的细胞的总数目/浓度。可选:标记的细胞与CellTracker或其他的非细胞毒性的标签为荧光显微镜。 </li><li>沉淀细胞，将沉淀重悬于5.0％的适当体积的PEG排除，以达到所需的细胞数。例如，一个96孔板需要37,500成纤维细胞重新悬浮在200μl的PEG产生合流翌日。适用于其他类型的细胞和文化基板尺寸缩放这些数字。 </li><li>使用微量移液器，免除0.5微升滴的DEX到干细胞培养基质。较大的体积的液滴产生较大的禁区。液滴大小不等，从0.1到1微升建议排除缩微。可选:DEX液滴可以沉积提前24小时的时间，并允许在室温下进行脱水。这可能会产生更清洁的模式。 </li><li>分配的200μl的PEG细胞悬液到吨他很好地掩饰的DEX液滴。 </li><li>在加湿培养箱中培养12小时，在37℃下，5％CO 2的地点。确保菜不倾斜，在处理过程中，它被放置在一个水平的培养箱搁板，以避免破坏模式。 </li><li>取出的PEG溶液，用200μl的培养基中洗涤三次。 </li><li>加入新鲜的培养基中，并返回到培养箱。 </li><li>定期监测的文化隔离区观察到细胞的运动。 </li></ol> 3。配置2:岛图案（96孔板格式） <ol><li>准备5.0％wt / wt的PEG和12.8％wt / wt的DEX在细胞培养基中，如上述的解决方案。 </li><li>收获细胞用于岛屿构图。确定可用的细胞的总数目/浓度。 </li><li>颗粒和重悬的细胞在适当体积的12.8％DEX岛屿构图以达到所需浓度的细胞。 5000行政长官的浓度LLS /μl以下建议强粘附性的细胞类型。对于细胞，具有难以附着或松散附着的细胞，最多至10,000个细胞/微升的浓度也可以考虑。 </li><li>工作迅速，以避免干燥，移液管加入0.5μl的DEX液滴到干细胞培养基材中，如上所述。不要让水滴干。可选:可以向孔中加入200μl的PEG溶液的时间提前。 DEX液滴，然后可以存入PEG的解决方案，在那里他们将沉入水底的文化联系面。这可能会产生更清洁的岛模式。 </li><li>量的DEX液滴用200μl的PEG。 </li><li>在加湿培养箱中培养12小时，在37℃下，5％CO 2的地点。确保菜不倾斜，在处理过程中，它被放置在一个水平的培养箱搁板，以避免破坏模式。 </li><li>取出的PEG溶液，用200μl的培养基中洗涤三次。 </li><li>添加新的培养介质和返回培养箱。 </li><li>定期监测的文化观察细胞运动和增殖向外的岛屿。 </li></ol> 4。配置3:排除文化（96孔板格式） <ol><li>准备5.0％wt / wt的PEG和12.8％wt / wt的DEX在细胞培养基中，如上述的解决方案。 </li><li>收获细胞用于排斥和岛屿构图。确定的总的数目/浓度的细胞，可用于不同的细胞类型。可选:有些细胞配对可能会显示增殖指数显着不同。为了防止被排除在外的的细胞overpopulating岛图案的细胞（尤其是长期文化），2小时排除与丝裂霉素C用于治疗的细胞，他们收获前或照射。这将防止扩散。来区分两种细胞群，如果必要，可以使用荧光CellTracker染料。 </li><li>颗粒细胞和​​悬浮颗粒exclusioŇ图案形成在适当体积的10.0％的PEG，以达到所需的细胞数，如上述。将沉淀重悬于岛图案形成在适当体积的12.8％DEX，以达到所需的浓度的细胞，如上述。 </li><li>使用微量移液器，免除0.5微升滴DEX细胞悬液到干细胞培养基质。不要让水滴干。 </li><li>量对DEX用200μl的PEG细胞悬浮液的液滴。 </li><li>在加湿培养箱中培养12小时，在37℃下，5％CO 2的地点。确保菜不倾斜，在处理过程中，它被放置在一个水平的培养箱搁板，以避免破坏模式。 </li><li>删除的PEG溶液，并在200μl的培养基中洗涤三次。 </li><li>加入新鲜的培养基中，并返回到培养箱。 </li><li>监视共培养，观察细胞群之间的相互作用。可选:可以控制排斥或岛屿的图案逐张进行准备idually，通过共培养细胞不发生相互作用，或通过阻断利益途径之前或之后，图案形成在一个或两个细胞群。 </li></ol>

<ol>
	<li>Hatti-Kaul, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aqueous+two-phase+systems+:+methods+and+protocols.">Aqueous two-phase systems : methods and protocols.</a> Methods in biotechnology. xiii, 440 (2000).</li><li>Albertsson, P. A. k. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Partition of cell particles and macromolecules: separation and purification of biomolecules, cell organelles, membranes, and cells in aqueous polymer two-phase systems and their use in biochemical analysis and biotechnology. , 346 (1986).</li><li>Yamada, M., 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=Continuous+cell+partitioning+using+an+aqueous+two-phase+flow+system+in+microfluidic+devices.">Continuous cell partitioning using an aqueous two-phase flow system in microfluidic devices.</a> Biotechnol. Bioeng. 88 (4), 489-494 (2004).</li><li>Soohoo, J. R., Walker, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+aqueous+two+phase+system+for+leukocyte+concentration+from+whole+blood.">Microfluidic aqueous two phase system for leukocyte concentration from whole blood.</a> Biomed. Microdevices. 11 (2), 323-329 (2009).</li><li>Hahn, T., Hardt, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+and+size+separation+of+DNA+samples+at+liquid-liquid+interfaces.">Concentration and size separation of DNA samples at liquid-liquid interfaces.</a> Anal. Chem. 83 (14), 5476-5479 (2011).</li><li>Hatti-Kaul, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aqueous+two-phase+systems.+A+general+overview.">Aqueous two-phase systems. A general overview.</a> Mol. Biotechnol. 19 (3), 269-277 (2001).</li><li>Hustedt, H., Kroner, K. H., Menge, U., Kula, M. -. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+recovery+using+two-phase+systems.">Protein recovery using two-phase systems.</a> Trends in Biotechnology. 3 (6), 139-144 (1985).</li><li>Keating, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aqueous+Phase+Separation+as+a+Possible+Route+to+Compartmentalization+of+Biological+Molecules.">Aqueous Phase Separation as a Possible Route to Compartmentalization of Biological Molecules.</a> Acc Chem. Res. 45 (12), 2114-2124 (2012).</li><li>Helfrich, M. R., 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=Partitioning+and+assembly+of+metal+particles+and+their+bioconjugates+in+aqueous+two-phase+systems.">Partitioning and assembly of metal particles and their bioconjugates in aqueous two-phase systems.</a> Langmuir. 21 (18), 8478-8486 (2005).</li><li>Diamond, A. D., Hsu, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prote.+Partitioning+in+PEG/Dextran+Aqueous+Two-Phase+Systems.">Prote. Partitioning in PEG/Dextran Aqueous Two-Phase Systems.</a> AIChE Journal. 36 (7), 1017-1024 (1990).</li><li>Y-T, Z. -. Q., Zhu, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+of+interfacial+tension+of+aqueous+two-phase+systems.">Modeling of interfacial tension of aqueous two-phase systems.</a> Chemical Engineering Science. 54 (4), 433-440 (1999).</li><li>Liu, Y., Lipowsky, R., Dimova, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+dependence+of+the+interfacial+tension+for+aqueous+two-phase+polymer+solutions+of+dextran+and+polyethylene+glycol.">Concentration dependence of the interfacial tension for aqueous two-phase polymer solutions of dextran and polyethylene glycol.</a> Langmuir. 28 (8), 3831-3839 (2012).</li><li>Rha, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interfacial+Tension+of+Polyethylene+Glycol/Potassium+Phosphate+Aqueous+Two-Phase+Systems.">Interfacial Tension of Polyethylene Glycol/Potassium Phosphate Aqueous Two-Phase Systems.</a> Physics and Chemistry of Liquids: An International Journal. 38 (1), 25-34 (2000).</li><li>Fang, 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=Rapid+Generation+of+Multiplexed+Cell+Cocultures+Using+Acoustic+Droplet+Ejection+Followed+by+Aqueous+Two-Phase+Exclusion+Patterning.">Rapid Generation of Multiplexed Cell Cocultures Using Acoustic Droplet Ejection Followed by Aqueous Two-Phase Exclusion Patterning.</a> Tissue Eng. Part C. Methods. 18 (9), 647-657 (2012).</li><li>Tavana, H., 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=Nanolitre+liquid+patterning+in+aqueous+environments+for+spatially+defined+reagent+delivery+to+mammalian+cells.">Nanolitre liquid patterning in aqueous environments for spatially defined reagent delivery to mammalian cells.</a> Nat. Mater. 8 (9), 736-741 (2009).</li><li>Tavana, H., Mosadegh, B., Takayama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymeric+aqueous+biphasic+systems+for+non-contact+cell+printing+on+cells:+engineering+heterocellular+embryonic+stem+cell+niches.">Polymeric aqueous biphasic systems for non-contact cell printing on cells: engineering heterocellular embryonic stem cell niches.</a> Adv. Mater. 22 (24), 2628-2631 (2010).</li><li>Tavana, H., 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=Microprinted+feeder+cells+guide+embryonic+stem+cell+fate.">Microprinted feeder cells guide embryonic stem cell fate.</a> Biotechnol. Bioeng. , (2011).</li><li>Tavana, H., Takayama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aqueous+biphasic+microprinting+approach+to+tissue+engineering.">Aqueous biphasic microprinting approach to tissue engineering.</a> Biomicrofluidics. 5 (1), 13404 (2011).</li><li>Frampton, J. P., 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=Precisely+targeted+delivery+of+cells+and+biomolecules+within+microchannels+using+aqueous+two-phase+systems.">Precisely targeted delivery of cells and biomolecules within microchannels using aqueous two-phase systems.</a> Biomed. Microdevices. 13 (6), 1043-1051 (2011).</li><li>Hossein Tavana, K. K., Bersano-Begey, T., Luker, K. E., Luker, G. D., Takayama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rehydration+of+Polymeric,+Aqueous,+Biphasic+System+Facilitates+High+Throughput+Cell+Exclusion+Patterning+for+Cell+Migration+Studies.">Rehydration of Polymeric, Aqueous, Biphasic System Facilitates High Throughput Cell Exclusion Patterning for Cell Migration Studies.</a> Advanced Functional Materials. 21 (15), 2920-2926 (2011).</li><li>Falconnet, 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=Surface+engineering+approaches+to+micropattern+surfaces+for+cell-based+assays.">Surface engineering approaches to micropattern surfaces for cell-based assays.</a> Biomaterials. 27 (16), 3044-3063 (2006).</li><li>Lim, J. Y., Donahue, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+sensing+and+response+to+micro-+and+nanostructured+surfaces+produced+by+chemical+and+topographic+patterning.">Cell sensing and response to micro- and nanostructured surfaces produced by chemical and topographic patterning.</a> Tissue Eng. 13 (8), 1879-1891 (2007).</li><li>Ringeisen, B. R., 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=Jet-based+methods+to+print+living+cells.">Jet-based methods to print living cells.</a> Biotechnol. J. 1 (9), 930-948 (2006).</li><li>Wright, 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=Generation+of+static+and+dynamic+patterned+co-cultures+using+microfabricated+parylene-C+stencils.">Generation of static and dynamic patterned co-cultures using microfabricated parylene-C stencils.</a> Lab Chip. 7 (10), 1272-1279 (2007).</li><li>Takayama, 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=Patterning+cells+and+their+environments+using+multiple+laminar+fluid+flows+in+capillary+networks.">Patterning cells and their environments using multiple laminar fluid flows in capillary networks.</a> Proc. Natl. Acad. Sci. U.S.A. 96 (10), 5545-5548 (1999).</li><li>Berthier, 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=Pipette-friendly+laminar+flow+patterning+for+cell-based+assays.">Pipette-friendly laminar flow patterning for cell-based assays.</a> Lab Chip. 11 (12), 2060-2065 (2011).</li><li>Davidson, R. L., O'Malley, K. A., Wheeler, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyethylene+glycol-induced+mammalian+cell+hybridization:+effect+of+polyethylene+glycol+molecular+weight+and+concentration.">Polyethylene glycol-induced mammalian cell hybridization: effect of polyethylene glycol molecular weight and concentration.</a> Somatic Cell Genet. 2 (3), 271-280 (1976).</li><li>Johnson, D. M., LaFranzo, N. A., Maurer, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creating+Two-Dimensional+Patterned+Substrates+for+Protein+and+Cell+Confinement.">Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement.</a> J. Vis. Exp. (55), e3164 (2011).</li><li>Moon, S., Lin, P., Keles, H. O., Yoo, S., Demirci, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Title+Cell+Encapsulation+by+Droplets.">Title Cell Encapsulation by Droplets.</a> J. Vis. Exp. (8), e316 (2007).</li></ol>

作者没有竞争的金融利益。

的ATPS细胞缩微方法只需要很少的细胞培养技术熟练的专业知识之外，可以很快掌握。这种方法的优点是，它是廉价的，快速的和具有多种类型的细胞和文化格式兼容。由于这些原因，我们的协议应该很容易通过生命科学家，特别是研究细胞的增殖，迁移和趋化性，近分泌和旁分泌的细胞群之间的相互作用的影响。这里介绍的分析，可以很容易地使用标准的图像分析程序，可用的软件，如ImageJ的细...

双水相的系统（ATPSs）形式时，两种不相容的聚合物的溶液混合在一起，在足够高的浓度。相分离的影响，由多种因素，其中包括的聚合物的分子量和极性，温度下的溶液，pH值和离子含量的水性溶剂1，2。在该点的两种聚合物溶液单独确定由所选择的三相系统的理化性质，但一般发生在低聚合物的浓度（小于20％重量/重量），在非变性条件下，允许将用于生物技术的ATPSs应用程序3-9。 到目前为止研究得最广泛的ATPS是聚乙二醇（PEG）/葡聚糖（DEX）系统。由这些廉价的和生物相容的聚合物形成的ATPS最初被描述为纯化的生物分子的分子的分区2，10的方式。分区没有贡献的相位系统的额外的分子或颗粒时，会发生混合与PEG和DEX。基于它们的相对亲和力地塞米松或PEG，分子或颗粒内的两个阶段中的一个或界面处将优先驻留。另一个特性PEG / DEX ATPS的是两种聚合物相之间的界面张力的存在。由PEG和DEX ATPSs形成一般显示是低得多的比其他如油和水的液-液两相体系的界面张力;然而，界面张力仍然发挥效果的小颗粒，如病毒，细胞和蛋白质总量2 11-13。最后，因为较高分子量的PEG和DEX单独的生理浓度的盐的存在下，在低浓度（小于5％的高分子量的聚合物品种的重量/重量），有一些对哺乳动物细胞的任何有害的影响，如果在这些注册成立系统14-16。 近日，界面性质和分区的ATPSs已被应用于本实验室的细胞图案14，16-20。这是通过缩微更密集地塞米松溶液对PEG的存在下，在细胞培养基材。当细胞被纳入PEG相，他们被排除进入对DEX由于PEG / DEX界面张力20的液滴。当细胞被图案化中的DEX相，它们被保持在通过界面张力和分区16，17，19的细胞培养基材的表面上。 在对比等方法进行细胞图案，ATPS的细胞图案是简单易学，只需要基本的知识有关的聚合物，并能进行细胞培养和使用微量移液器。细胞图案的其他方法往往涉及专门的设备和培训，不容易被转换为日e生活科学。例如，一些方法（微接触印刷或喷墨印刷）图案细胞间接通过施加模式的细胞粘附的生物分子到培养基材，随后作为电池附件21，22的网站。尽管间接方法对于某些类型的细胞是有用的，它们需要用户的技能和专门的设备来制造图案形成工具的高度的，并且可以取决于对特定类型的细胞/生物分子图案缺乏特异性。此外，细胞可以沉积模式特异性高的方式直接图形的方法，包括层流图案，制版和喷墨印刷23日至26日 。然而，这些技术也需要用户的专业知识和专门的设备，在印刷过程中，可能会损坏细胞。尽管这些方法通常会产生的细胞，细胞图案的精确模式是一个有用的工具，在生命科学领域，它必须是符合成本效益的一个实现ND简单。 在这里，我们报告的详细描述在我们之前发布的应用程序使用ATPSs产生图案的细胞培养协议。只使用微量移液器，用户可以生成细胞的禁区或细胞的岛屿迁移实验。这是通过PEG / DEX的界面张力，可以保持细胞在对DEX相中或排除细胞沉积在从DEX PEG相方式。通过梳理上述两个基本图案形成技术，它是可能的，以迅速产生如肝成纤维细胞共培养的细胞的共培养。图案形成方法，ATPS参数和预期结果进行详细说明。

<table border="1"><tbody><tr><td> 试剂 </td><td> 生产厂家 </td></tr><tr><td>右旋糖酐50万kDa的</td><td> Pharmacosmos，丹麦</td></tr><tr><td>聚乙二醇35,000 kDa的</td><td> Sigma-Aldrich公司，圣路易斯，密苏里州</td></tr><tr><td> Hela细胞</td><td> ATCC，弗吉尼亚州马纳萨斯</td></tr><tr><td> HepG2细胞C3A </td><td> ATCC，弗吉尼亚州马纳萨斯</td></tr><tr><td> NIH 3T3 </td><td> ATCC，弗吉尼亚州马纳萨斯</td></tr><tr><td>手机追踪器</td><td> Invitrogen公司，卡尔斯巴德，加利福尼亚州</td></tr><tr><td> DMEM </td><td> Gibco公司，卡尔斯巴德，加利福尼亚州</td></tr><tr><td> RPMI </td><td> Gibco公司，卡尔斯巴德，加利福尼亚州</td></tr><tr><td> F12 </td><td> Gibco公司，卡尔斯巴德，加利福尼亚州</td></tr><tr><td>胎牛血清</td><td> Gibco公司，卡尔斯巴德，加利福尼亚州</td></tr></tbody></table>

cell co-culture patterning using aqueous two-phase systems

细胞图案化技术，速度快，易于使用，价格合理的高通量细胞分析平台的未来发展将需要研究细胞间的相互作用和组织工程化系统。此详细的协议描述了一种用于产生的细胞的共培养物，使用生物相容的解决方案的葡聚糖（DEX）和聚乙二醇（PEG），发生相分离时，在阈值以上的浓度相结合的方法。细胞可以使用此方法的配置的各种图案。可以通过印刷在基板上的液滴的DEX和覆盖它们的PEG含有细胞的溶液进行细胞排斥构图。两者之间的聚合物溶液形成的界面张力，使细胞落在对DEX液滴周围的外侧，并形成了一个环形的结算可以用于迁移实验。细胞岛可以分配到的PEG溶液的细胞丰富的DEX阶段，或图案覆盖的DEX液滴与PEG的溶液。共培养物，可形成直接与地塞米松岛屿构图通过结合细胞排斥。这些方法兼容的各种液体处理方法，包括手动微穿刺，可用于几乎任何粘附的细胞类型。

要选择PEG和DEX细胞图案的适当组合，重要的是要确定的双结点曲线。这条曲线描绘的点，可以形成一个ATPS可以改变为一组给定的基础上的温度，pH值和离子含量的聚合物。用于培养细胞的需要定制的培养基配方中，可能有必要通过实验确定的双结点曲线。这是通过产生一系列ATPSs是远离的双结点和在他们的PEG和DEX内容变化（ 图1，紫色圆圈）。 ATPS是本时，聚合物溶液出现混浊混合时?...

Watch this Scientific Journal Video about Cell Co-culture Patterning Using Aqueous Two-phase Systems at JoVE.com

Cell Co-culture Patterning Using Aqueous Two-phase Systems

双水相系统，用于同时图案多个的细胞群体。此快速和容易的方法，细胞图案利用葡聚糖和聚​​乙二醇的水溶液，和两者之间的聚合物溶液中存在的界面张力的相分离。

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-co-culture-patterning-using-aqueous-two-phase-systems

Research

JoVE Journal

Bioengineering

18.3K 次观看.  University of Michigan. 双水相系统，用于同时图案多个的细胞群体。此快速和容易的方法，细胞图案利用葡聚糖和聚​​乙二醇的水溶液，和两者之间的聚合物溶液中存在的界面张力的相分离。

视频: Cell Co-culture Patterning Using Aqueous Two-phase Systems

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. ...

A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The toolkit consists of multiple standard interchangeable parts (BioBricks)9 addressing the conversion of alkanes, regulation of gene expression and survival in toxic hydrocarbon-rich environments.
A three-step pathway for alkane degradation was implemented in E. coli to enable the conversion of medium- and long-chain alkanes to their respective alkanols, alkanals and ultimately alkanoic-acids. The latter were metabolized via the native &beta;-oxidation pathway. To facilitate the oxidation of medium-chain alkanes (C5-C13) and cycloalkanes (C5-C8), four genes (alkB2, rubA3, rubA4and rubB) of the alkane hydroxylase system from Gordonia sp. TF68,21 were transformed into E. coli. For the conversion of long-chain alkanes (C15-C36), theladA gene from Geobacillus thermodenitrificans was implemented. For the required further steps of the degradation process, ADH and ALDH (originating from G. thermodenitrificans) were introduced10,11. The activity was measured by resting cell assays. For each oxidative step, enzyme activity was observed.
To optimize the process efficiency, the expression was only induced under low glucose conditions: a substrate-regulated promoter, pCaiF, was used. pCaiF is present in E. coli K12 and regulates the expression of the genes involved in the degradation of non-glucose carbon sources.
The last part of the toolkit - targeting survival - was implemented using solvent tolerance genes, PhPFD&alpha; and &beta;, both from Pyrococcus horikoshii OT3. Organic solvents can induce cell stress and decreased survivability by negatively affecting protein folding. As chaperones, PhPFD&alpha; and &beta; improve the protein folding process e.g. under the presence of alkanes. The expression of these genes led to an improved hydrocarbon tolerance shown by an increased growth rate (up to 50%) in the presences of 10% n-hexane in the culture medium were observed.
Summarizing, the results indicate that the toolkit enables E. coli to convert and tolerate hydrocarbons in aqueous environments. As such, it represents an initial step towards a sustainable solution for oil-remediation using a synthetic biology approach.

A sustainable auto regulating bacterial system for the remediation of oil pollutions was designed using standard interchangeable DNA parts (BioBricks). An engineered E. coli strain was used to degrade alkanes via &beta;-oxidation in toxic aqueous environments. The respective enzymes from different species showed alkane degradation activity. Additionally, an increased tolerance to n-hexane was achieved by introducing genes from alkane-tolerant bacteria.

一个工具包，使水环境中的烃类转化

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The ...

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 ...

Robotic Production of Cancer Cell Spheroids with an Aqueous Two-phase System for Drug Testing

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing both mono-culture and co-culture spheroids in a high throughput 96-well plate format is described in this work. This approach utilizes an aqueous two-phase system to confine cells into a drop of the denser aqueous phase immersed within the second aqueous phase. The drop rests on the well surface and keeps cells in close proximity to form a single spheroid. This technology has been adapted to a robotic liquid handler to produce size-controlled spheroids and expedite the process of spheroid production for compound screening applications. Spheroids treated with a clinically-used drug show reduced cell viability with increase in the drug dose. The use of a standard micro-well plate for spheroid generation makes it straightforward to analyze viability of cancer cells of drug-treated spheroids with a micro-plate reader. This technology is straightforward to implement both robotically and with other liquid handling tools such as manual pipettes.

A protocol for robotic printing of cancer cell spheroids in a high throughput 96-well plate format using an aqueous two-phase system is presented.

癌症细胞球体机器人生产与药物测试的双水相系统

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing ...

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

Organic contaminants adsorbed on the surface of titanium dioxide (TiO2) can be decomposed by photocatalysis under ultraviolet (UV) light. Here we describe a novel protocol employing the TiO2 photocatalysis to locally alter cell affinity of the substrate surface. For this experiment, a thin TiO2 film was sputter-coated on a glass coverslip, and the TiO2 surface was subsequently modified with an organosilane monolayer derived from octadecyltrichlorosilane (OTS), which inhibits cell adhesion. The sample was immersed in a cell culture medium, and focused UV light was irradiated to an octagonal region. When a neuronal cell line PC12 cells were plated on the sample, cells adhered only on the UV-irradiated area. We further show that this surface modification can also be performed in situ, i.e., even when cells are growing on the substrate. Proper modification of the surface required an extracellular matrix protein collagen to be present in the medium at the time of UV irradiation. The technique presented here can potentially be employed in patterning multiple cell types for constructing coculture systems or to arbitrarily manipulate cells under culture.

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

We describe a protocol for modifying cell affinity of a scaffold surface in aqueous environment. The method takes advantage of titanium dioxide photocatalysis to decompose organic film in the photo-irradiated region. We show that it can be used to create microdomains of scaffolding proteins, both ex situ and in situ.

光图案蛋白质和细胞中的水环境中使用二氧化钛<sub&gt; 2</sub&gt;光催化

Organic contaminants adsorbed on the surface of titanium dioxide (TiO2) can be decomposed by photocatalysis under ultraviolet (UV) light. Here we describe ...

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 ...

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

分层海藻酸钠构造:一个异质细胞群的共培养平台

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

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 ...

利用双水相系统的细胞共培养图案

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