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>

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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?...

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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 ...

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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.