作者要感谢克里斯·莫罗的技术援助。经费是由美国国立卫生研究院SBIR（R43）提供给予1R43GM101859-01（MPI）GRANT10940803。

1细胞培养制备<ol><li>存储NIH/3T3细胞的股票在液氮中，直到准备使用。 </li><li>准备完整的媒体使用Dulbecco改良的Eagle培养基（DMEM），补充有10％胎牛血清，10mM的HEPES缓冲液，50单位/ ml青霉素和50μg/ ml的链霉素NIH/3T3细胞。 </li><li>在37°C解冻细胞2-3分钟的水浴摇床</li><li>将细胞再悬浮于5ml完全培养基和离心机在1500×g离心3分钟。 </li><li>除去细胞上清液而不干扰细胞沉淀。 </li><li>重悬细胞于5ml介质，并使用血细胞计数器计数细胞。 <ol><li>在0.5 ml离心管中取出10微升的细胞悬液和地点。 </li><li>加10微升台盼蓝的细胞悬浮液，并搅拌用移液管的尖端。 </li><li>移液管加入10μl染色细胞成血细胞计数室。 </li><li>计数细胞，血球在10倍放大倍率。 <ol><li>计数活细胞（白色小球）为4的9大广场。只计算，不会出现从台盼蓝染色深蓝色的活细胞。 </li><li>计算每个大正方形单元的平均数目，从而导致细胞的原始细胞悬液×10 4个细胞/ ml的数量。 </li><li>种子细胞以1×10 5个细胞/毫升，总的5毫升在每个T25烧瓶的密度。 </li></ol></li><li>培养的细胞在开始实验前70％至80％汇合之间。刷新媒体每2-3天或根据需要。 </li></ol></li></ol> 2，印刷表面处理<ol><li>马克在组织培养处理的聚苯乙烯（TCTPS）12孔板的底部，标记一个矩形的打印头（7毫米x 19毫米）的大小。 </li><li>移液管加入50μl胎牛血清成的矩形区域，并铺在其整个区域的小费。 </li><li>离开培养皿在无菌生物安全罩O / N，使血清斑点完全干燥。这些板块应使用不超过2天前编制。 </li></ol> 3，浸入式印刷<ol><li>清洗打印头用蒸馏水。 <ol><li>填60毫米培养皿中，用蒸馏水和停靠打印头在表面上。 </li><li>使用气动泵以150微升/分钟，通过每行的流动蒸馏水2分钟。 </li><li>从培养皿倒掉水，并用干净的水填满它。 </li><li>移动打印头向上和向下在水中的3倍。 </li></ol></li><li>停靠在打印头中心多年来在预先制备的12孔板中的血清涂覆点。 </li><li>总理打印头与预热的完全培养基，每通道300微升。 </li><li>以50000个细胞/ ml的浓度在表面上和60微升/分钟，每通道100微升打印悬浮细胞。 </li><li>将打印头，离开了停靠反对表面上，并且在细胞培养孵化器歧管设置为5％的CO 2和37℃下进行2小时。 </li><li>在2小时培养后，取出打印头，然后将盖子上的培养皿。在该打印头上取下的时候被认为是0小时的时间点。 </li></ol> 4，标准的细胞培养<ol><li>加入1 ml预热的媒介，在步骤2中制备的血清斑TCTPS 12孔板中的一个。 </li><li>取1ml所剩余的细胞悬浮液的步骤2和吸管成12孔TCTPS板的单个孔。这会产生含50,000个细胞在培养皿的文化。 </li><li>将细胞培养板在培养箱中在37℃下搅拌2小时。 </li><li>经过2小时培养开始计时;用于印刷和播种样品之间的一致性，这被认为是0小时时间点。 </li></ol>五，文化可视化<ol><li> 0,2，24，和48小时取单元从所述两个方法d时旁切上面和图像使用标准的倒置显微镜。 <ol><li>使用碘化丙啶，红色荧光染色，是只掺入死细胞的细胞核染色的细胞。 <ol><li>轻轻冲洗细胞3次，用1ml磷酸盐缓冲盐水中钙和镁（PBS + +），以除去任何碎片。 </li><li>加入1 ml预热的PBS + +的每个培养容器。 </li><li>加入1μl的碘化丙啶溶液（1毫克/毫升），以每个培养容器。 </li><li>孵育染色用碘化丙啶，在37℃下进行10分钟的细胞。 </li></ol></li><li>图片使用在10倍倒置显微镜和40倍的放大倍率的细胞。 </li></ol></li><li>丢弃在适当的生物危害容器中的细胞。 </li></ol>

<ol>
	<li>Fernandes, T. G., Diogo, M. M., Clark, D. S., Dordick, J. S., Cabral, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+cellular+microarray+platforms:+applications+in+drug+discovery,+toxicology+and+stem+cell+research.">High-throughput cellular microarray platforms: applications in drug discovery, toxicology and stem cell research.</a> Trends in Biotechnology. 27, 342-349 (2009).</li><li>Michelini, E., Cevenini, L., Mezzanotte, L., Coppa, A., Roda, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-based+assays:+fuelling+drug+discovery.">Cell-based assays: fuelling drug discovery.</a> Anal Bioanal Chem. 398, 227-238 (2010).</li><li>Gao, 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=Recent+developments+in+microfluidic+devices+for+in+vitro+cell+culture+for+cell-biology+research.">Recent developments in microfluidic devices for in vitro cell culture for cell-biology research.</a> TrAC Trends in Analytical Chemistry. 35, 150-164 (2012).</li><li>Geysen, H. M., Schoenen, F., Wagner, D., Wagner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+compound+libraries+for+drug+discovery:+an+ongoing+challenge.">Combinatorial compound libraries for drug discovery: an ongoing challenge.</a> Nature Reviews Drug Discovery. 2, 222-230 (2003).</li><li>Xu, Y., Shi, Y., Ding, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+approach+to+stem-cell+biology+and+regenerative+medicine.">A chemical approach to stem-cell biology and regenerative medicine.</a> Nature. 453, 338-344 (2008).</li><li>Khademhosseini, A., Langer, R., Borenstein, J., Vacanti, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscale+technologies+for+tissue+engineering+and+biology.">Microscale technologies for tissue engineering and biology.</a> Proceedings of the National Academy of Sciences of the United States of America. 103, 2480-2487 (2006).</li><li>Bhadriraju, K., Chen, 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=Engineering+cellular+microenvironments+to+improve+cell-based+drug+testing.">Engineering cellular microenvironments to improve cell-based drug testing.</a> Drug Discovery Today. 7, 612-620 (2002).</li><li>Castel, D., Pitaval, A., Debily, M. -. A., Gidrol, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+microarrays+in+drug+discovery.">Cell microarrays in drug discovery.</a> Drug Discov. Today. 11, 616-622 (2006).</li><li>Tsui, J. H., Lee, W., Pun, S. H., Kim, J., Kim, D. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics-assisted+in+vitro+drug+screening+and+carrier+production.">Microfluidics-assisted in vitro drug screening and carrier production.</a> Advanced Drug Delivery Reviews. , (2013).</li><li>Gottwald, 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=A+chip-based+platform+for+the+in+vitro+generation+of+tissues+in+three-dimensional+organization.">A chip-based platform for the in vitro generation of tissues in three-dimensional organization.</a> Lab on a Chip. 7, 777-785 (2007).</li><li>Liu, R., Lee, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Integrated Biochips for DNA Analysis. , (2008).</li><li>Natarajan, 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=Continuous-flow+microfluidic+printing+of+proteins+for+array-based+applications+including+surface+plasmon+resonance+imaging.">Continuous-flow microfluidic printing of proteins for array-based applications including surface plasmon resonance imaging.</a> Analytical Biochemistry. 373, 141-146 (2008).</li><li>Natarajan, S., Hatch, A., Myszka, D. G., Gale, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+Conditions+for+Protein+Array+Deposition+Using+Continuous+Flow.">Optimal Conditions for Protein Array Deposition Using Continuous Flow.</a> Anal. Chem. 80, 8561-8567 (2008).</li><li>Keighley, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+need+for+high+throughput+kinetics+early+in+the+drug+discovery+process.">The need for high throughput kinetics early in the drug discovery process.</a> , (2011).</li><li>Xu, 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=A+three-dimensional+in+vitro+ovarian+cancer+coculture+model+using+a+high-throughput+cell+patterning+platform.">A three-dimensional in vitro ovarian cancer coculture model using a high-throughput cell patterning platform.</a> Biotechnol J. 6, 204-212 (2011).</li><li>Tuckwell, D. S., Weston, S. A., Humphries, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrins:+a+review+of+their+structure+and+mechanisms+of+ligand+binding.">Integrins: a review of their structure and mechanisms of ligand binding.</a> Symposia of the Society for Experimental Biology. 47, 107 (1993).</li><li>Roberts, 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=Using+mixed+self-assembled+monolayers+presenting+RGD+and+(EG)+3OH+groups+to+characterize+long-term+attachment+of+bovine+capillary+endothelial+cells+to+surfaces.">Using mixed self-assembled monolayers presenting RGD and (EG) 3OH groups to characterize long-term attachment of bovine capillary endothelial cells to surfaces.</a> Journal of the American Chemical Society. 120, 6548-6555 (1998).</li><li>Chandra, R. A., Douglas, E. S., Mathies, R. A., Bertozzi, C. R., Francis, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmable+cell+adhesion+encoded+by+DNA+hybridization.">Programmable cell adhesion encoded by DNA hybridization.</a> Angewandte Chemie. 118, 910-915 (2006).</li><li>Hsiao, S. 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=Direct+cell+surface+modification+with+DNA+for+the+capture+of+primary+cells+and+the+investigation+of+myotube+formation+on+defined+patterns.">Direct cell surface modification with DNA for the capture of primary cells and the investigation of myotube formation on defined patterns.</a> Langmuir. 25, 6985-6991 (2009).</li><li>Black, C. B., Duensing, T. D., Trinkle, L. S., Dunlay, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-Based+Screening+Using+High-Throughput+Flow+Cytometry.">Cell-Based Screening Using High-Throughput Flow Cytometry.</a> ASSAY and Drug Development Technologies. 9, 13-20 (2011).</li><li>Derby, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioprinting:+inkjet+printing+proteins+and+hybrid+cell-containing+materials+and+structures.">Bioprinting: inkjet printing proteins and hybrid cell-containing materials and structures.</a> J. Mater. Chem. 18, 5717-5721 (2008).</li><li>Onoe, 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=Cellular+Microfabrication:+Observing+Intercellular+Interactions+Using+Lithographically-Defined+DNA+Capture+Sequences.">Cellular Microfabrication: Observing Intercellular Interactions Using Lithographically-Defined DNA Capture Sequences.</a> Langmuir. 28, 8120-8126 (2012).</li><li>Hsiao, S. 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=DNA-Coated+AFM+Cantilevers+for+the+Investigation+of+Cell+Adhesion+and+the+Patterning+of+Live+Cells.">DNA-Coated AFM Cantilevers for the Investigation of Cell Adhesion and the Patterning of Live Cells.</a> Angewandte Chemie International Edition. 47, 8473-8477 (2008).</li></ol>

作者是瓦萨奇微流体的员工和股东产生试剂和/或本手稿中使用的仪器。

这里所描述的3D微流体印刷技术是唯一能够细胞microfluidically印刷阵列放入盛好液体， 即一个水下表面。通过印刷在潜面，细胞微阵列可以制造维持细胞的生理相关的细胞表型，以及多路复用单元中的单个孔图4底部的能力。本研究的结果表明，microfluidically印刷细胞的结果在细胞附着与细胞相媲美的形态和活力;然而，印刷单元可被更密集地附着在导致较短的总研究时间定义的位?...

在医药工业中的最新进展已导致使用移动芯片在药物发现过程用于药物筛选和cytotoxicological分析1,2,3兴趣增加。利用细胞微阵列体外高通量检测和筛选方法的发展将有利于候选药物的快速和具有成本效益的发展，以及提前1,4单元的基本认识。传统的方法来筛查细胞采用常规孔板平台;然而，这种方法由于成本高，产量有限，以及对细胞功能1,5定量信息有限，能力有限。由于这些限制，研究在细胞微阵列技术是新兴的分子生物学特性，组织工程和药物筛选1,6。细胞微阵列的优点包括更小的样品使用，影响最小细胞表型异质性掩蔽的信息，最重要的自动化测定法更高通量应用1,7,8的能力。 制药行业目前采用高通量细胞系筛选测定与2D细胞单层培养物在微量滴定孔板9的药物筛选。复细胞在微量滴定板的孔中提供了独特的实验方案更高的吞吐量的潜力。另外，在目前的技 ​​术用于蜂窝芯片使细胞干这可能显着地从改变细胞的表型在体内 10,11。为了克服这些问题，MFCA被设计并且被示于图1。的MFCA三维流体的设计使打印头前端在图1中被降低到一个浴和压靠在一个表面上以形成一个密封。打印头的软硅胶尖的行​​为就像一个垫片，形成了一个可逆的密封。该MFCA技术是唯一适合用潜面，这都需要细胞培养和组织切片系统的接口，并且是很难或不可能与大多数其他方法。销或喷墨印刷将无法正常工作，和2D的微流体装置不适合于沉积或离散点的大型阵列接口。另外，通过小型化和定位的实验 - 细胞微阵列 - 的MFCA克服了与高通量细胞系筛选测定法相关的主要问题。 该CFM采用3D频道网络周期小体积流体样品在表面上的微观12,13点的位置。通过用流量进行打印时，生物分子，细胞和其它试剂均保持在一个液体环境中在整个印刷过程中，使敏感的生物分子和细胞的印刷不暴露于空气中，这妨碍了当前小区的印刷技术。另外，也可以直接从粗物质，如杂交瘤或提供上清液有该阵列表面上的捕获机构进行打印。该原稿的目的是详细浸没印刷两种细胞类型的解释在表面上。

<table border="0" cellpadding="0" cellspacing="0" width="571">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Continuous Flow Microspotter</td>
			<td style="width:71px;">Wasatch Microfluidics</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">NIH/3T3 cells</td>
			<td style="width:71px;">ATCC</td>
			<td style="width:119px;">CRL-1658</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Dubbleco&#39;s Modified Eagle Medium</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">11965-092</td>
			<td>base media for cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">HEPES buffer</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">15630-080</td>
			<td>cell media additive (control pH)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Sodium pyruvate</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">11360-070</td>
			<td>cell media additive</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Penicillin-Streptomycin&#160;</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;"></td>
			<td>cell media additive</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Trypan blue</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">15250-061</td>
			<td>stain cell sfor counting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Haemocytometer</td>
			<td style="width:71px;">Fisher</td>
			<td align="right" style="width:119px;">267110</td>
			<td>cell chamber to count cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Nikon Eclipse TS100</td>
			<td style="width:71px;">Nikon</td>
			<td style="width:119px;"></td>
			<td>Used to check on cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Nikon Eclipse TE2000-U</td>
			<td style="width:71px;">Nikon</td>
			<td style="width:119px;"></td>
			<td>Used for collecting images</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Phosphate Buffered Saline (with calcium and magnesium)</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">14040-133</td>
			<td>rinsing cells before passaging and before staining with PI</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">TrypLE Express&#160;</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">A12177-01</td>
			<td>used to remove cells from surface</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;"></td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

the submerged printing of cells onto a modified surface using a continuous flow microspotter

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell phenotype after printing, poor organization and distribution of desired cells, and the inability to deliver drugs and/or nutrients to targeted areas in the array. Our 3D microfluidic printing technology is uniquely capable of sealing and printing arrays of cells onto submerged surfaces in an automated and multiplexed manner. The design of the microfluidic cell array (MFCA) 3D fluidics enables the printhead tip to be lowered into a liquid-filled well or dish and compressed against a surface to form a seal. The soft silicone tip of the printhead behaves like a gasket and is able to form a reversible seal by applying pressure or backing away. Other cells printing technologies such as pin or ink-jet printers are unable to print in submerged applications. Submerged surface printing is essential to maintain phenotypes of cells and to monitor these cells on a surface without disturbing the material surface characteristics. By printing onto submerged surfaces, cell microarrays are produced that allow for drug screening and cytotoxicity assessment in a multitude of areas including cancer, diabetes, inflammation, infections, and cardiovascular disease.

的成纤维细胞系NIH/3T3细胞被印刷或接种到水下表面。使细胞生长至每毫升5×10 4个细胞的密度。细胞被用传统的细胞培养技术接种。细胞使用大幅面，12 - 流动池的打印头，其中的信道是较大的（〜500微米）比用于蛋白质和其他生物分子的CFM打印。将细胞印刷或接种在血清涂覆的表面。印刷过程示于图1。 印刷及接种后，将细胞可视化，以评估密度和形貌?...

Watch this Scientific Journal Video about The Submerged Printing of Cells onto a Modified Surface Using a Continuous Flow Microspotter at JoVE.com

The Submerged Printing of Cells onto a Modified Surface Using a Continuous Flow Microspotter

This 3D microfluidic printing technology prints arrays of cells onto submerged surfaces. We describe how arrays of cells are delivered microfluidically in 3D flow cells onto submerged surfaces. By printing onto submerged surfaces, cell microarrays were produced that allow for drug screening and cytotoxicity assessment in a multitude of areas.

细胞的浸入式印刷到改性表面采用连续流动Microspotter

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell ...

the-submerged-printing-cells-onto-modified-surface-using-continuous

Research

JoVE Journal

Bioengineering

8.6K 次观看.  Wasatch Microfluidics. This 3D microfluidic printing technology prints arrays of cells onto submerged surfaces. We describe how arrays of cells are delivered microfluidically in 3D flow cells onto submerged surfaces. By printing onto submerged surfaces, cell microarrays were produced that allow for drug screening and cytotoxicity assessment in a multitude of areas.

视频: The Submerged Printing of Cells onto a Modified Surface Using a Continuous Flow Microspotter

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses1.
Our flow chamber design and flow circuit (Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs2,3.
This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts.
The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (&gt; 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry (Fig. 11E), or scanning electron microscopy5. The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12)6.

We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.

平行板流动腔和评估内皮祖细胞层流剪应力下连续流电路

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well ...

科研

生物工程

Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using an automated robotic dispensing system. The particular bioink was selected as it allows cells to sediment towards and sense the features. The dispensing system bioprints viable cells in hydrogel bioinks using a backpressure assisted print head. However, by replacing the print head with a sharpened stylus or scalpel, the dispensing system can also be employed to create topographical cues through surface etching. The stylus movement can be programmed in steps of 10 &#181;m in the X, Y and Z directions. The patterned grooves were able to orientate mesenchymal stem cells, influencing them to adopt an elongated morphology in alignment with the grooves&#39; direction. The patterning could be designed using plotting software in straight lines, concentric circles, and sinusoidal waves. In a subsequent procedure, fibroblasts and mesenchymal stem cells were suspended in a 2% gelatin bioink, for bioprinting in a backpressure driven extrusion printhead. The cell bearing bioink was then printed using the same programmed coordinates used for the etching. The bioprinted cells were able to sense and react to the etched features as demonstrated by their elongated orientation along the direction of the etched grooves.

This protocol describes a bioprinting methodology using an automated robotic depositing system that incorporates etched topographical guidance cues with the precision deposition of a cell bearing hydrogel bioink. The printed cells are directly delivered to the etched features and are able to sense and orientate with them.

自动点胶机器人技术在细胞表面和指导生物印刷

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using ...

Gene Expression Analysis of Endothelial Cells Exposed to Shear Stress Using Multiple Parallel-plate Flow Chambers

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate flow chambers. Endothelial cells form the inner cellular lining of blood vessels and are chronically exposed to the frictional force of blood flow called shear stress. Under physiological conditions, endothelial cells function in the presence of various shear stress conditions. Thus, the application of shear stress conditions in in vitro models can provide greater insight into endothelial responses in vivo. The parallel-plate flow chamber previously published by Lane et al.9 is adapted to study endothelial gene regulation in the presence and absence of steady (non-pulsatile) laminar flow. Key adaptations in the set-up for laminar flow as presented here include a large, dedicated environment to house concurrent flow circuits, the monitoring of flow rates in real-time, and the inclusion of an exogenous reference RNA for the normalization of quantitative real-time PCR data. To assess multiple treatments/conditions with the application of shear stress, multiple flow circuits and pumps are used simultaneously within the same heated and humidified incubator. The flow rate of each flow circuit is measured continuously in real-time to standardize shear stress conditions throughout the experiments. Because these experiments have multiple conditions, we also use an exogenous reference RNA that is spiked-in at the time of RNA extraction for the normalization of RNA extraction and first-strand cDNA synthesis efficiencies. These steps minimize the variability between samples. This strategy is employed in our pipeline for the gene expression analysis with shear stress experiments using the parallel-plate flow chamber, but parts of this strategy, such as the exogenous reference RNA spike-in, can easily and cost-effectively be used for other applications.

Here, a workflow for the culture and gene expression analysis of endothelial cells under fluid shear stress is presented. Included is a physical arrangement for simultaneously housing and monitoring multiple flow chambers in a controlled environment and the use of an exogenous reference RNA for quantitative PCR.

多平行板流室对剪切应力暴露的内皮细胞基因表达分析

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate ...

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and introduces uncertainty in results due to variability between scorers. To remedy this, automated slide-scanning microscopy as well as conventional flow cytometry methods have been introduced in an attempt to remove scorer bias and improve throughput. However, these methods have their own inherent limitations such as inability to visualize the cytoplasm of the cell and the lack of visual MN verification or image data storage with flow cytometry. Multispectral Imaging Flow Cytometry (MIFC) has the potential to overcome these limitations. MIFC combines the high resolution fluorescent imagery of microscopy with the statistical robustness and speed of conventional flow cytometry. In addition, all collected imagery can be stored in dose-specific files. This paper describes the protocol developed to perform a fully automated version of the MN assay on MIFC. Human lymphoblastoid TK6 cells were enlarged using a hypotonic solution (75 mM KCl), fixed with 4% formalin and the nuclear content was stained with Hoechst 33342. All samples were run in suspension on the MIFC, permitting acquisition of high resolution images of all key events required for the assay (e.g. binucleated cells with and without MN as well as mononucleated and polynucleated cells). Images were automatically identified, categorized and enumerated in the MIFC data analysis software, allowing for automated scoring of both cytotoxicity and genotoxicity. Results demonstrate that using MIFC to perform the in vitro MN assay allows statistically significant increases in MN frequency to be detected at several different levels of cytotoxicity when compared to solvent controls following exposure of TK6 cells to Mitomycin C and Colchicine, and that no significant increases in MN frequency are observed following exposure to Mannitol.

The in vitro micronucleus assay is a well-established method for evaluating genotoxicity and cytotoxicity but scoring the assay using manual microscopy is laborious and suffers from subjectivity and inter-scorer variability. This paper describes the protocol developed to perform a fully automated version of the assay using multispectral imaging flow cytometry.

使用多光谱成像流细胞测定法执行体外微核测定的自动化方法

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process starts by screening for colonies that survive on minimal media, assuming that only Escherichia coli harboring the correct plasmid will be able to thrive in the specific conditions. Since the process of building large genetic circuits, requiring the assembly of many DNA parts, is challenging, circuit components are often distributed between multiple plasmids at different copy numbers requiring the use of several antibiotics. Mutations in the plasmid can destroy transcription of the antibiotic resistance genes and interject with resources management in the cell leading to necrosis. The selected colony is set on a glass-bottom Petri dish and a few focus planes are selected for microscopy tracking in both bright field and fluorescent domains. The protocol maintains the image focus for more than 12 hours under initial conditions that cannot be regulated, creating a few difficulties. For example, dead cells start to accumulate in the lenses' field of focus after a few hours of imaging, which causes toxins to buildup and the signal to blur and decay. Depletion of nutrients introduces new metabolic processes and hinder the desired response of the circuit. The experiment's temperature lowers the effectivity of inducers and antibiotics, which can further damage the reliability of the signal. The minimal media gel shrinks and dries, and as a result the optical focus changes over time. We developed this method to overcome these challenges in Escherichia coli, similar to previous works developing analogous methods for other micro-organisms. In addition, this method offers an algorithm to quantify the total stochastic noise in unaltered and altered cells, finding that the results are consistent with flow analyzer predictions as shown by a similar coefficient of variation (CV).

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

This work presents a microscopy method that allows live imaging of a single cell of Escherichia coli for analysis and quantification of the stochastic behavior of synthetic gene circuits.

使用延时显微镜连续测量 大肠杆菌 中的生物噪声

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process ...

The Fabrication and Operation of a Continuous Flow, Micro-Electroporation System with Permeabilization Detection

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is accompanied by high manufacturing costs, which has brought about an interest in using alternative methods for gene delivery. Electroporation is an electro-physical, non-viral approach for the intracellular delivery of genes and other exogenous materials. Upon the application of an electric field, the cell membrane temporarily allows molecular delivery into the cell. Typically, electroporation is performed on the macroscale to process large numbers of cells. However, this approach requires extensive empirical protocol development, which is costly when working with primary and difficult-to-transfect cell types. Lengthy protocol development, coupled with the requirement of large voltages to achieve sufficient electric-field strengths to permeabilize the cells, has led to the development of micro-scale electroporation devices. These micro-electroporation devices are manufactured using common microfabrication techniques and allow for greater experimental control with the potential to maintain high throughput capabilities. This work builds off a microfluidic-electroporation technology capable of detecting the level of cell membrane permeabilization at a single-cell level under continuous flow. However, this technology was limited to 4 cells processed per second, and thus a new approach for increasing the system throughput is proposed and presented here. This new technique, denoted as cell-population-based feedback control, considers the cell permeabilization response to a variety of electroporation pulsing conditions and determines the best-suited electroporation pulse conditions for the cell type under test. A higher-throughput mode is then used, where this 'optimal' pulse is applied to the cell suspension in transit. The steps for fabricating the device, setting up and running the microfluidic experiments, and analyzing the results are presented in detail. Finally, this micro-electroporation technology is demonstrated by delivering a DNA plasmid encoding for green fluorescent protein (GFP) into HEK293 cells.

This protocol describes the microfabrication techniques required to build a lab-on-a-chip, microfluidic electroporation device. The experimental setup performs controlled, single-cell-level transfections in a continuous flow and can be extended to higher throughputs with population-based control. An analysis is provided showcasing the ability to electrically monitor the degree of cell membrane permeabilization in real-time.

具有透化检测功能的连续流动微电穿孔系统的制造和操作

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is ...

Fabrication of Engineered Vascular Flaps Using 3D Printing Technologies

Engineering implantable, functional, thick tissues requires designing a hierarchical vascular network. 3D bioprinting is a technology used to create tissues by adding layer upon layer of printable biomaterials, termed bioinks, and cells in an orderly and automatic manner, which allows for creating highly intricate structures that traditional tissue engineering techniques cannot achieve. Thus, 3D bioprinting is an appealing in vitro approach to mimic the native vasculature complex structure, ranging from millimetric vessels to microvascular networks. 
Advances in 3D bioprinting in granular hydrogels enabled the high-resolution extrusion of low-viscosity extracellular matrix-based bioinks. This work presents a combined 3D bioprinting and sacrificial mold-based 3D printing approach for fabricating engineered vascularized tissue flaps. 3D bioprinting of endothelial and support cells using recombinant collagen-methacrylate bioink within a gelatin support bath is utilized for the fabrication of a self-assembled capillary network. This printed microvasculature is assembled around a mesoscale vessel-like porous scaffold, fabricated using a sacrificial 3D printed mold, and is seeded with endothelial cells. 
This assembly induces the endothelium of the mesoscale vessel to anastomose with the surrounding capillary network, establishing a hierarchical vascular network within an engineered tissue flap. The engineered flap is then directly implanted by surgical anastomosis to a rat femoral artery using a cuff technique. The described methods can be expanded for the fabrication of various vascularized tissue flaps for use in reconstruction surgery and vascularization studies.

Engineered flaps require an incorporated functional vascular network. In this protocol, we present a method of fabricating a 3D printed tissue flap containing a hierarchical vascular network and its direct microsurgical anastomoses to rat femoral artery.

使用3D打印技术制造工程血管瓣

Engineering implantable, functional, thick tissues requires designing a hierarchical vascular network. 3D bioprinting is a technology used to create ...

Automation of the Micronucleus Assay Using Imaging Flow Cytometry and Artificial Intelligence

The micronucleus (MN) assay is used worldwide by regulatory bodies to evaluate chemicals for genetic toxicity. The assay can be performed in two ways: by scoring MN in once-divided, cytokinesis-blocked binucleated cells or fully divided mononucleated cells. Historically, light microscopy has been the gold standard method to score the assay, but it is laborious and subjective. Flow cytometry has been used in recent years to score the assay, but is limited by the inability to visually confirm key aspects of cellular imagery. Imaging flow cytometry (IFC) combines high-throughput image capture and automated image analysis, and has been successfully applied to rapidly acquire imagery of and score all key events in the MN assay. Recently, it has been demonstrated that artificial intelligence (AI) methods based on convolutional neural networks can be used to score MN assay data acquired by IFC. This paper describes all steps to use AI software to create a deep learning model to score all key events and to apply this model to automatically score additional data. Results from the AI deep learning model compare well to manual microscopy, therefore enabling fully automated scoring of the MN assay by combining IFC and AI.

The micronucleus (MN) assay is a well-established test for quantifying DNA damage. However, scoring the assay using conventional techniques such as manual microscopy or feature-based image analysis is laborious and challenging. This paper describes the methodology to develop an artificial intelligence model to score the MN assay using imaging flow cytometry data.

使用成像流式细胞术和人工智能实现微核检测自动化

The micronucleus (MN) assay is used worldwide by regulatory bodies to evaluate chemicals for genetic toxicity. The assay can be performed in two ways: by ...

用于连续 DLP 打印的生物墨水特性的定量表征

Quantitative Characterization of Liquid Photosensitive Bioink Properties for Continuous Digital Light Processing Based Printing

Precise printing fabrication of bioinks is a prerequisite for tissue engineering; the Jacobs working curve is the tool to determine the precise printing parameters of digital light processing (DLP). However, the acquisition of working curves wastes materials and requires high formability of materials, which are not suitable for biomaterials. In addition, the reduction of cell activity due to multiple exposures and the failure of structural formation due to repeated positioning are both unavoidable problems in conventional DLP bioprinting. This work introduces a new method of obtaining the working curve and the improvement process of continuous DLP printing technology based on such a working curve. This method of obtaining the working curve is based on the absorbance and photorheological properties of the biomaterials, which do not depend on the formability of the biomaterials. The continuous DLP printing process, obtained from improving the printing process by analyzing the working curve, increases the printing efficiency more than tenfold and greatly improves the activity and functionality of cells, which is beneficial to the development of tissue engineering.

作者聚焦：基于连续数字光处理的印刷中液体光敏生物墨水特性的定量表征  

This study uses temperature and material composition to control the yield stress properties of yield stress fluids. The solid-like state of the ink can protect the printing structure, and the liquid-like state can continuously fill the printing position, realizing the digital light processing 3D printing of extremely soft bioinks.

基于连续数字光处理的印刷中液体光敏生物墨水特性的定量表征

Precise printing fabrication of bioinks is a prerequisite for tissue engineering; the Jacobs working curve is the tool to determine the precise printing ...