Kind gift of THP-1 cells from Dr. Mark Chong and assistance in microfabrication from Dr. Yuejun Kang and Dr. Nishanth V. Menon (School of Chemical and Biomedical Engineering, Nanyang Technological University) were greatly acknowledged. This project was funded by NTU-Northwestern Institute of Nanomedicine (Nanyang Technological University). H.W.H. was supported by Lee Kong Chian School of Medicine (LKCMedicine) postdoctoral fellowship.

间充质干细胞和单核细胞的纳米1.（NPS）标签<ol><li>在Dulbecco改良的Eagle培养基（DMEM）培养间充质干细胞（MSCs）补充有10％胎牛血清（FBS）和抗生素，以≥80％汇合标记之前。类似地，培养的THP-1细胞（ATCC）在罗斯韦尔园区纪念研究所（RPMI）1640培养基中补充有10％FBS的至〜10 6个细胞/ ml的密度。 </li><li>负载二氧化硅纳米粒子（〜500微米）用搅拌一夜钙黄绿素染料溶液（200微米）。制造使用前述协议PLGA-AM钙黄绿素（CAM）。22 <ol><li>溶解250微克的CAM和100mg PLGA（50:50）的氯仿，在4℃。 </li><li>生成在室温下使用高速均化器（13600 XG，60秒）的单乳剂纳米粒。使用离心分离（3400×g离心5分钟），洗涤（双分销商蒸发在收集之前的化学罩（≥3小时）的氯仿LLED水），冷冻干燥和储存于-20℃。 </li></ol></li><li>孵育CAM-PLGA纳米颗粒（1毫克），或钙黄绿素的二氧化硅纳米粒子（150微克）在0.01％聚-L-赖氨酸（PLL），在室温下溶液15 - 20分钟。 </li><li>离心机在3400×g离心5分钟以在1毫升各自培养基的纳米颗粒再悬浮之前除去过量的PLL上清液。 </li><li>孵育细胞（MSC或THP-1，〜1 - 2×10 6个细胞在总）的纳米颗粒约24小时（0.1毫克·毫升-1贴标浓度）。 </li><li>分离并收获使用2毫升0.25％胰蛋白酶（5分钟，37℃）的标记的粘附MSC和用6ml DMEM中淬灭。在细胞（1000 xg离心，4分钟），并悬浮降速至10 5的浓度- 10 6个细胞/毫升的微流体的处理。使用标记的THP-1细胞（10月5日至10月6日细胞/ ml）直接用于微流体的净化。 </li></ol> 2.微流体器件的制备<ol><li> 器件制造 <ol><li>制造微流体螺旋装置（500微米（W）×115微米（h））进行与来自使用标准软光刻步骤的商业试剂盒聚二甲基硅氧烷（PDMS）。23 </li><li>拌30 G碱基预聚物和3克在称量舟彻底固化剂。解气体中60分钟的干燥器将混合物以除去任何气泡。 </li><li>倒入PDMS混合到与螺旋通道设计图案的硅片模具师傅细心的〜5的高度 - 10mm以下。 </li><li>解气体在干燥器真空60分钟将混合物再次以除去任何气泡。重复这个过程，直到所有的气泡被淘汰。 </li><li>固化在80℃的炉中的PDMS混合物2小时，直到PDMS被设置。确保在晶片的固化具有恒定装置高度期间不倾斜。 </li><li>剪下用手术刀PDMS螺旋装置并小心剥离从母模的PDMS板。 </li><li> TR即时通讯装置的边缘用手术刀以确保表面平滑以便键合。 </li><li>冲床入口两个孔（1.5毫米），两洞（1.5 mm）适用使用1.5毫米活检冲床的PDMS设备上的插座。 </li><li>用异丙醇（IPA）冲洗设备以除去任何碎片和干燥设备在80℃的烘箱中5分钟。 </li><li>清洁使用胶带将PDMS设备（与通道功能）的底面。 </li><li>用遮蔽胶带的载玻片（2"3"）的清洁一侧。 </li><li>小心放置和暴露PDMS设备和载玻片的清理过的表面在等离子体清洁器的腔室，并使其受到真空60秒。接下来，打开等离子最大功率，直到室变成了粉红色的颜色降低室内的压力。 <ol><li>暴露表面以空气等离子体60秒。当进入物理等离子体创建的PDMS和玻璃使紧粘结的暴露表面上反应性物质人接触。关闭等离子体功率和释放来自等离子体清洁器的压力来检索设备和载玻片。 </li></ol></li><li>通过紧紧按压暴露于等离子体的表面，并确保没有气泡被困在两个表面之间粘合的PDMS设备和载玻片在一起。 </li><li>加热使用加热板2小时设定在80℃，以加强键合的键合设备。 </li></ol></li><li> 设备操作 <ol><li>油管切割两片（1.52毫米外径）的约15 - 20厘米对进口注射器和每个管子的一端固定一个注射器尖端（表23）。 </li><li>切管两片（1.52毫米外径）的〜5 - - 10厘米为网点和附加到PDMS设备的出口孔。 </li><li>事先，直到其流出的出口管的样品运行，手动素用含70％乙醇的注射器设备。允许乙醇坐30秒至1分钟来灭菌装置。 </li><li>负载30毫升过滤（0.2微米孔径）鞘缓冲液（磷酸盐缓冲盐水（PBS）中含有0.1％牛血清白蛋白（BSA））转换成60毫升注射器和固定在注射器泵的注射器。将泵的正确设置（注射器尺寸:60毫升，容量60,000微升）。 注:加入的BSA的PBS中是最小化的细胞 - 细胞的结合和非特异性细胞和PDMS设备之间的结合。 </li><li>负载3毫升标记细胞进3毫升注射器并固定在一个单独的注射泵的注射器。将泵的正确设置（注射器尺寸:3毫升，体积:3000微升）。 </li><li>检查没有气泡被截留在注射器以确保稳定的流动。轻轻喷射液体滴出，喷嘴去除气泡。 </li><li>入口注射器提示和管路连接到注射器，并把它们插入到设备（鞘和样品入口）各自的入口。保证有沿管子无气泡。 </li><li>安装设备到一个反转在细胞分选过程中编相位差显微镜即时成像。 </li><li>安全小烧杯浪费和两个15毫升管靠近设备上使用粘合剂的显微镜阶段。 </li><li>放置出口管路进入废物烧杯中。 </li><li>对样品鞘缓冲流动比率设置为1:10并同时启动注射泵启动排序过程（例如:120微升/ min的样品注射器和1200微升/ min的护套的注射器;渠道流速〜0.38米/秒）。 </li><li>运行设备1.5分钟的流量稳定。这可以通过在明场通道内壁附近惯性聚焦细胞存在相衬使用高速摄像机进行确认（5000〜 - 每10,000个帧（fps），曝光时间:10-50微秒）。 </li><li>定位的出口管路成不同的管，以从细胞出口和废弃物出口收集洗脱液。 </li></ol></li></ol>

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本文所描述的DFF细胞纯化技术使以高通量的方式标记的细胞的快速和连续分离。这种分离方法非常适合于大样本容量或高浓度的细胞样本处理，比传统的基于膜过滤是容易长时间使用后堵塞更好。同样，基于亲和力的磁选需要这是费力又昂贵的附加细胞标记的步骤。纯化的细胞被示为在通道内保持它们的标记，由于停留时间短剂和细胞形态以及（ 图3）以最小流量/剪切诱导的作用（剪切应力τ，〜200-250达因/厘米2）（&lt; 1秒）20,24。此外，用户可以通过简单地代与所需介质或缓冲溶液鞘缓冲液（盐水）选择排序标记的细胞的洗脱溶液中。 这是至关重要的有一个在微通道之前使用它可以细胞集中行为产生不利的影响没有灰尘或碎屑。这可以通过彻底用IPA清洗入口孔，以及使用掩蔽胶带清洁等离子体接合之前与PDMS表面来实现。由于微型高流量条件下运行（〜1 - 1.5毫升/分钟），它也是重要的加热在电炉上键合的PDMS装置，确保与PDMS和载玻片之间的强粘接。在器件操作中，如果细胞不聚焦良好，这意味着流率是不充分的。人们可以通过检查设备的入口区域和注射器提示，以确保没有泄漏故障排除。 从标记细胞绑定纳米粒子的去除是不是因为常见的实验室分离技术（如离心）琐碎不能单独颗粒/不同大小（ 例如，未结合的颗粒和标记细胞）细胞。尽管人们可以争辩说，反复清洗和培养基的变化可以去除从贴壁细胞游离纳米颗粒在一定程度上，这个过程是费力的和无效的悬浮细胞培养物。该DFF微流体装置利用基于大小的排序和是高度通用的，因为它可以同时用于悬浮和贴壁细胞具有大尺寸范围，以及去除不同的材料的NP的。如果靶细胞是不同的尺寸，所述DFF设备可以通过改变流动条件或通道几何结构（ 例如，通道高度以及内和外通道宽度的比率），以实现有效的分离进行优化。先前已表明，相比于在纳米粒子除去具有最小细胞损失常规离心DFF优越。22此外，细胞恢复性能仍然很高（&gt; 90％）时的样品浓度增加到10 7个细胞/ ml。这是在现有的惯性微流体细胞分离方法这是一般的关键改进LY限于10 5个细胞/毫升容量由于细胞-细胞相互作用和细胞在平衡位置过分拥挤。25 而DFF技术实现基于大小差异的分离，它不能被用于其它细胞工程方法如标记和未标记的细胞的分离，由于在小区大小可忽略的变化。对于基于亲和力的排序和其它常用的分离方式（磁化率和导电率）的细胞表面标记物，可考虑这样的潜在的应用。 DFF技术也并不适合基于荧光多路复用细胞分选。一个更好的选择是使用荧光激活细胞为标记的细胞与在高吞吐量操作不同的荧光的单步排序分选（FACS），但需要昂贵和笨重的设备。 最后，该协议是用于工程细胞时的纯化是非常有用的含变革剂（生长因子，药物，皮质类固醇等 ）纳米粒子被用于生物成像和/或细胞疗法。这是因为残留的NP自由能发挥上， 在体内注射时对这些外来颗粒响应邻近细胞或免疫细胞脱靶变革效应。它也可能会无意中产生信号生物成像过程中可能会产生误导。之前使用这些游离纳米粒子的除去从而风险降低到非靶细胞，最小化在免疫系统汇编，并减少细胞的连续标记/工程中的步骤之间的污染的问题。其成功的实施大大方便细胞标记，使在治疗基于粒子细胞工程的无干扰的使用情况。

由代理加载微/纳米颗粒（纳米颗粒）的工程细胞是一种简单的，基因组整合-自由，和通用的方法，以提高生物成像能力和增加/再生医学补充其天然的治疗性质。1-3蜂窝修改都通过标记所获得的质膜或细胞质剂加载纳米粒的过量浓度以饱和结合位点。然而，这种方法的主要缺点是留在溶液中后细胞标记过程未结合颗粒的显著数量时，其可以潜在地混淆颗粒工程细胞的精确识别或4,5-复杂化的治疗效果。此外，接触含变革纳米粒在浓度过高剂（生长因子，皮质类固醇等 ）可导致细胞毒性和误导暴露可引起对非靶细胞意想不到的后果。甚至包括o颗粒载体F"生物相容性"的材料[ 例如 ，聚（乳酸共乙醇酸），PLGA]可以煽动在某些条件下，以及有效的免疫细胞反应。6这与免疫功能受损（ 例如 ，类风湿性关节炎），其潜在的延迟个体特别危险全身纳米间隙。7因此，现有的引入颗粒的工程细胞的高效去除游离颗粒是非常重要的，以减少毒性和降低误导暴露于体内剂加载颗粒。 常规梯度离心常被用来改造的细胞从游离颗粒中分离，但是费力的和在批处理模式操作。此外，通过高速离心期间细胞和密度梯度介质可能损害细胞的完整性和/或影响细胞行为的组分经历剪切应力。8微流体是与SEV一个有吸引力的替代方案ERAL分离技术，包括确定性的横向位移（DLD）9，dieletrophoresis 10,11和acoustophoresis为小颗粒分离和缓冲交换应用开发12。然而，这些方法从低通量遭受（1-10微升·分钟- 1），并容易堵塞的问题。活性分离如基于介电电泳的方法还需要在固有的介电电泳细胞表型或其他细胞标记的步骤来实现的分离的差异。一个更有前景的方法包括惯性微流体-颗粒或细胞的横向迁移跨越简化集中在由于在高雷诺数（Re）13显性升力（F L）不同的位置由于其高流动条件和优越的尺寸分辨率。 ，它经常被利用的规模为基础的细胞分离14,15和缓冲交换应用。16-18 HoweveR，缓冲液交换性能仍然很差与~10-30％污染物的溶液，作为分离的细胞通常保持接近原始和新的缓冲液之间的边界。16-18更重要的是，靶细胞的大小分布是相似的实现精确惯性聚焦和分离从其中提出了一个问题特别是在异质大小的细胞类型，如间充质干细胞（MSC）的处理的原缓冲溶液19中我们先前已开发出一种新的惯性微流体细胞分选技术称为迪安流分离（DFF），用于使用一个2入口，2-插座螺旋微器件隔离循环肿瘤细胞（的CTC）从全血20和细菌21。在此视频协议中，我们将描述标签的THP-1（人急性单核细胞白血病细胞系）的悬浮液单核细胞（〜15微米）和干细胞（10-30微米）用钙黄绿素-L的方法oaded纳米粒，其次是DFF螺旋微型为标记的细胞和除去未纳米粒的有效回收的制造和操作。22这种单一步骤纯化策略使标记的悬浮液，悬浮于新鲜缓冲液中没有离心粘附细胞连续恢复。此外，它可以处理高达10万个细胞·毫升-1，细胞密度为顺应再生医学应用。

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			<td colspan="4" height="21" style="height:21px;width:780px;">Cell lines &#38; Media</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Mesenchymal Stem Cells (MSCs)</td>
			<td style="width:127px;">Lonza</td>
			<td style="width:119px;">PT-2501</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)</td>
			<td style="width:127px;">Lonza</td>
			<td style="width:119px;">12-614F</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:119px;">10270-106</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:368px;">THP-1 monocyte cells (THP-1)</td>
			<td style="width:127px;">ATCC</td>
			<td style="width:119px;">TIB-202</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Roswell Park Memorial Institute (RPMI) 1640 media</td>
			<td style="width:127px;">Lonza</td>
			<td style="width:119px;">12-702F</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Name</td>
			<td style="width:127px;">Company</td>
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			<td>Comments/Description</td>
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			<td colspan="4" height="21" style="height:21px;width:780px;">Reagents &#38; Materials</td>
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			<td height="21" style="height:21px;width:368px;">0.01% poly-L-lysine (PLL)</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:119px;">P8920</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:368px;">3 mL Syringe</td>
			<td style="width:127px;">BD</td>
			<td style="width:119px;">302113</td>
			<td>Syringe 3ml Luer-Lock</td>
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			<td style="width:127px;">BD</td>
			<td style="width:119px;">309653</td>
			<td>Syringe 60ml Luer-Lock</td>
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			<td height="21" style="height:21px;width:368px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:127px;">Biowest</td>
			<td style="width:119px;">P6154-100GR</td>
			<td></td>
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			<td height="21" style="height:21px;width:368px;">Calcein, AM (CAM)</td>
			<td style="width:127px;">Life Technologies</td>
			<td style="width:119px;">C1430</td>
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			<td height="21" style="height:21px;width:368px;">Calcein</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:119px;">C0875</td>
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			<td height="21" style="height:21px;width:368px;">Isopropanol</td>
			<td style="width:127px;">Fisher Chemical</td>
			<td style="width:119px;">#P/7507/17</td>
			<td>HPLC Grade 2.5 L</td>
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			<td height="21" style="height:21px;width:368px;">Phosphate-Buffered Saline (PBS)</td>
			<td style="width:127px;">Lonza</td>
			<td style="width:119px;">17-516Q/12</td>
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			<td height="21" style="height:21px;width:368px;">Plain Microscope Slides</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td style="width:119px;">FIS#12-550D</td>
			<td>75 X 25 X 1 mm</td>
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			<td height="21" style="height:21px;width:368px;">Polydimethylsiloxane (PDMS)</td>
			<td style="width:127px;">Dow Corning</td>
			<td style="width:119px;">SYLGARD&#174; 184</td>
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			<td height="21" style="height:21px;width:368px;">Scotch tape</td>
			<td style="width:127px;">3M</td>
			<td style="width:119px;">21200702044</td>
			<td>18mm x 25m</td>
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			<td height="21" style="height:21px;width:368px;">Silica NPs (&#8764;200 &#956;m)</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:119px;">748161</td>
			<td>Pore size 4 nm</td>
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			<td height="21" style="height:21px;width:368px;">Syringe Tip</td>
			<td style="width:127px;">JEC Technology</td>
			<td style="width:119px;">7018302</td>
			<td style="width:167px;">23GA .013 X .25</td>
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			<td height="21" style="height:21px;width:368px;">Trypsin-EDTA (0.25%)</td>
			<td style="width:127px;">Life Technologies</td>
			<td style="width:119px;">25200-056</td>
			<td></td>
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			<td height="21" style="height:21px;width:368px;">Tygon Tubing</td>
			<td style="width:127px;">Spectra-Teknik</td>
			<td style="width:119px;">06419-01</td>
			<td>.02X.06&#34; 100</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:368px;">Name</td>
			<td style="width:127px;">Company</td>
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			<td>Comments/Description</td>
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			<td colspan="4" height="21" style="height:21px;width:780px;">Equipment</td>
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			<td height="21" style="height:21px;width:368px;">Biopsy punch</td>
			<td style="width:127px;">Harris Uni-Core</td>
			<td>69036-15</td>
			<td>1.50 mm</td>
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			<td height="21" style="height:21px;width:368px;">Dessicator</td>
			<td style="width:127px;">Scienceware</td>
			<td style="width:119px;">111/4 IN OD</td>
			<td></td>
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			<td height="21" style="height:21px;width:368px;">High-speed Camera</td>
			<td style="width:127px;">Phantom&#160;</td>
			<td style="width:119px;">V9.1</td>
			<td></td>
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			<td height="21" style="height:21px;width:368px;">Inverted phase-contrast microscope&#160;</td>
			<td style="width:127px;">Nikon</td>
			<td>Eclipse Ti</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:368px;">Plasma cleaner</td>
			<td style="width:127px;">Harrick Plasma</td>
			<td style="width:119px;">PDC-002</td>
			<td></td>
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			<td height="21" style="height:21px;width:368px;">Syringe Pump</td>
			<td style="width:127px;">Chemyx</td>
			<td style="width:119px;">CX Fusion 200</td>
			<td></td>
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microfluidic buffer exchange for interference-free micro/nanoparticle cell engineering

与活性成分载微/纳米工程细胞（NPS）正在成为一个日益流行的方法，以提高本地治疗特性，使生物成像和控制细胞表型。一个关键尚未充分解决的问题是，仍无法通过常规的离心容易地除去细胞标记后未结合颗粒的显著数目。这导致增加的生物成像的背景噪声，并且可以赋予变革效应到相邻的非靶细胞。在这个协议中，我们提出称为院长流分离（DFF）惯性微流体为基础的缓冲区交换策略，以高通量的方式免费纳米粒子标记细胞有效地分离。发达螺旋微型装置便于悬浮在新的缓冲溶液纯化的细胞（THP-1和干细胞）的连续集合（&gt; 90％细胞回收），同时实现未结合的荧光染料或染料加载纳米粒（SIL的&gt; 95％的耗尽ICA或PLGA）。这个单步，基于大小的细胞纯化策略使高细胞处理吞吐量（10 6个细胞/分），并且是微/纳米工程细胞大容积细胞纯化以实现无干扰的临床应用是非常有用的。

标记与生物显像剂装载纳米粒过夜细胞后，将标记的细胞（含有自由粒子）收获并通过DFF螺旋微型纯化以除去游离的NP在单一步骤的过程（ 图1A）。该2入口，2出口螺旋微通道是由工程软件设计和使用微制造SU-8光刻胶。然后将图案化的硅晶片被用作用于使用软光刻技术（ 图1B）的PDMS复制品成型的模板。来执行细胞分选，而内壁入口运行在一个较高的流速（1:10）的新鲜缓冲溶液捏细胞样品流动细胞样品泵入螺旋微型装置的外壁的入口。分离是通过惯性聚焦较大标记的细胞在内壁和朝向外壁（ 图2A）小未结合的纳米粒子的迪安-引起的回流来实现。该器件是第一APPLI编辑从装有荧光钙黄绿素纳米颗粒二氧化硅悬浮标记细胞的单核细胞（THP-1）进行排序（〜大小500纳米）。通过高速摄像（ 图2A），为确认和流式细胞术分析（ 图2B），获得较高的分离效率。从设备细胞出口收集洗脱液的图像还表明有效地除去二氧化硅和PLGA纳米颗粒 - 从THP-1细胞（〜1 5微米）。此外，荧光成像显示，在标记的THP-1细胞内化的NP DFF纯化（插图）（ 图3）中被很好地保留。因为MSC大小异质性，螺旋微通道的几何形状随后通过改变通道的高度和分岔设计，以使干细胞（ 图4）的有效收集改性。 <img alt="图1" src="/files/ftp_upload/54327/54327fig1.jpg" /> 图1（A）OVerall工作流程，利用院长流分离（DFF）微流控技术的悬架和贴壁细胞后快速标签单步​​微/纳米粒子（NPS）的去除。 （B）中的DFF螺旋装置由工程软件设计并使用微加工的硅晶片上形成图案。 PDMS设备由软光刻方法以后产生。实验装置由2个注射泵注入标记细胞在不同的流量转化为持续的净化和标记细胞的采集设备的新鲜生理盐水缓冲液中。 <a href="https://www.jove.com/files/ftp_upload/54327/54327fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54327/54327fig2.jpg" /> 图2. 纳米粉体通过DFF标记THP-1细胞去除。（A）的插图DFF分离原理。较大的标记细胞出现强劲的惯性升力（F L，红色箭头）和Dean阻力（F D，黄色箭头），并专注于微通道内壁附近。较小的纳米粒和缓冲溶液仅受迪安阻力和再循环到外壁达到分离。代表性的高速和表示THP-1细胞（红色箭头）和二氧化硅纳米粒子在出口区域装载有钙黄绿素染料的平衡位置的荧光图像。基于流式细胞术通过门控大小（FSC-A）和粒度（SSC-A）特性所决定的分析（B）的分离性能。 <a href="https://www.jove.com/files/ftp_upload/54327/54327fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54327/54327fig3.jpg" /> 图3。 <strong<html> &gt;的纯化的标记使用DFF。插图显示相应的相位合同（上图）和单个细胞的荧光显微镜图像THP-1细胞（红色箭头）和NPS去除率代表亮场图像。 <a href="https://www.jove.com/files/ftp_upload/54327/54327fig3large.jpg" target="_blank">请点击此处查看这个大版本数字。</a> <img alt="图4" src="/files/ftp_upload/54327/54327fig4.jpg" /> 图4.从纳米粒子间充质干细胞（MSCs）通过DFF。（A）代表高速图像说明干细胞的有效分离（红色箭头）和PLGA纳米粒（蓝色箭头）到不同网点的去除 。黄色虚线指示微通道壁的大致位置。 （B）分离干细胞从二氧化硅和PLGA纳米粒性能。PG"目标="_空白"&gt;点击此处查看该图的放大版本。

Watch this Scientific Journal Video about Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering at JoVE.com

Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

这个协议描述了使用惯性基于微流体缓冲交换策略的纯化微/纳米工程细胞与未结合的颗粒的有效消耗。

微流控缓冲液更换为无干扰微/纳米颗粒细胞工程

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic ...

microfluidic-buffer-exchange-for-interference-free-micronanoparticle

Nanyang Technological University

Research

JoVE Journal

Bioengineering

9.0K 次观看.  Nanyang Technological University. 这个协议描述了使用惯性基于微流体缓冲交换策略的纯化微/纳米工程细胞与未结合的颗粒的有效消耗。 

视频: Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

Site-specific Bacterial Chromosome Engineering: &#934;C31 Integrase Mediated Cassette Exchange (IMCE)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as plasmid replication, plasmid stability, plasmid incompatibility, and plasmid copy number variance. This method uses the site-specific integrase from the Streptomyces phage (&Phi;) C312,3. The &Phi;C31 integrase catalyzes a direct recombination between two specific DNA sites: attB and attP (34 and 39 bp, respectively)4. This recombination is stable and does not revert5. A "landing pad" (LP) sequence consisting of a spectinomycin- resistance gene, aadA (SpR), and the E. coli &szlig;-glucuronidase gene (uidA) flanked by attP sites has been integrated into the chromosomes of Sinorhizobium meliloti, Ochrobactrum anthropi, and Agrobacterium tumefaciens in an intergenic region, the ampC locus, and the tetA locus, respectively. S. meliloti is used in this protocol. Mobilizable donor vectors containing attB sites flanking a stuffer red fluorescent protein (rfp) gene and an antibiotic resistance gene have also been constructed. In this example the gentamicin resistant plasmid pJH110 is used. The rfp gene6 may be replaced with a desired construct using SphI and PstI. Alternatively a synthetic construct flanked by attB sites may be sub-cloned into a mobilizable vector such as pK19mob7. The expression of the &Phi;C31 integrase gene (cloned from pHS628) is driven by the lac promoter, on a mobilizable broad host range plasmid pRK78139.
A tetraparental mating protocol is used to transfer the donor cassette into the LP strain thereby replacing the markers in the LP sequence with the donor cassette. These cells are trans-integrants. Trans-integrants are formed with a typical efficiency of 0.5%. Trans-integrants are typically found within the first 500-1,000 colonies screened by antibiotic sensitivity or blue-white screening using 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X-gluc). This protocol contains the mating and selection procedures for creating and isolating trans-integrants.

A quick and efficient method to integrate foreign DNA of interest into pre-made acceptor strains, termed landing pad strains, is described. The method allows site-specific integration of a DNA cassette into the engineered landing pad locus of a given strain, through conjugation and expression of the &#934;C31 integrase.

网站特定的细菌染色体工程:ΦC​​31整合介导的盒式交换（IMCE）

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as ...

科研

生物工程

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

研究蛋白质折叠的微流体混合器

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of interest following intravenous or intraarterial infusion. Consequently, increasing cell homing is currently studied as a strategy to improve cell therapy. Cell rolling on the vascular endothelium is an important step in the process of cell homing and can be probed in-vitro using a parallel plate flow chamber (PPFC). However, this is an extremely tedious, low throughput assay, with poorly controlled flow conditions. Instead, we used a multi-well plate microfluidic system that enables study of cellular rolling properties in a higher throughput under precisely controlled, physiologically relevant shear flow1,2. In this paper, we show how the rolling properties of HL-60 (human promyelocytic leukemia) cells on P- and E-selectin-coated surfaces as well as on cell monolayer-coated surfaces can be readily examined. To better simulate inflammatory conditions, the microfluidic channel surface was coated with endothelial cells (ECs), which were then activated with tumor necrosis factor-&#945; (TNF-&#945;), significantly increasing interactions with HL-60 cells under dynamic conditions. The enhanced throughput and integrated multi-parameter software analysis platform, that permits rapid analysis of parameters such as rolling velocities and rolling path, are important advantages for assessing cell rolling properties in-vitro. Allowing rapid and accurate analysis of engineering approaches designed to impact cell rolling and homing, this platform may help advance exogenous cell-based therapy.

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

This study used a multi-well plate microfluidic system, significantly increasing throughput of cell rolling studies under physiologically relevant shear flow. Given the importance of cell rolling in the multi-step cell homing cascade and the importance of cell homing following systemic delivery of exogenous populations of cells in patients, this system offers potential as a screening platform to improve cell-based therapy.

的系统分析<em&gt;体外</em&gt;细胞滚动使用多孔板微流控系统

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

电池挤压作为一种强大的，微流控细胞内传送平台

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~102 cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

微流体技术，探讨细胞变形

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

Controlled Microfluidic Environment for Dynamic Investigation of Red Blood Cell Aggregation

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white blood cells and platelets that are suspended in blood plasma. Due to the abundance of the RBCs (40% to 45% of the blood volume), their behavior dictates the rheological behavior of blood especially in the microcirculation. At very low shear rates, RBCs are seen to assemble and form entities called aggregates, which causes the non-Newtonian behavior of blood. It is important to understand the conditions of the aggregates formation to comprehend the blood rheology in microcirculation. The protocol described here details the experimental procedure to determine quantitatively the RBC aggregates in microcirculation under constant shear rate, based on image processing. For this purpose, RBC-suspensions are tested and analyzed in 120 x 60 &#181;m poly-dimethyl-siloxane (PDMS) microchannels. The RBC-suspensions are entrained using a second fluid in order to obtain a linear velocity profile within the blood layer and thus achieve a wide range of constant shear rates. The shear rate is determined using a micro Particle Image Velocimetry (&#181;PIV) system, while RBC aggregates are visualized using a high speed camera. The videos captured of the RBC aggregates are analyzed using image processing techniques in order to determine the aggregate sizes based on the images intensities.

The protocol described details an experimental procedure to quantify Red Blood Cell (RBC) aggregates under a controlled and constant shear rate, based on image processing techniques. The goal of this protocol is to relate RBC aggregate sizes to the corresponding shear rate in a controlled microfluidic environment.

微流控控制环境的红细胞聚集动态调查

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white ...

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 ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

高通量单细胞的分离和文化的微流体平台

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

多级可变高度的光刻多层带瓣微流体装置

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.