Name: micropatterning and assembly of 3d microvessels
Uploaded: 2016-09-09T15:00:00.000+00:00
Duration: 13 min 5 s
Description: Watch this Scientific Journal Video about Micropatterning and Assembly of 3D Microvessels at JoVE.com

笔者想承认Lynn和迈克·加维成像实验室的研究所干细胞与再生医学，以及在华盛顿大学华盛顿纳米加工设施。他们也承认美国国立卫生研究院的资助授予DP2DK102258（YZ到），以及培训补助T32EB001650（SSK来和MAR）和T32HL007312（以MAR）。

1.图案聚二甲基硅氧烷（PDMS）与网络设计的微细<ol><li> 晶圆制造到创造的网络设计的负模板 <ol><li>创建使用任何计算机辅助设计（CAD）软件的网络格局。确保入口和出口之间的对角线尺寸匹配在未来的步骤（见2.1.1）的壳体装置的入口和出口储槽之间的距离。 注意:本身是定义这取决于用户的具体的研究目标的图案的设计（ 见图1，例如图案）。 </li><li>生成使用高分辨率打印的网络图案的面罩或从商业供应商订购了镀铬光罩。光刻工艺的更详细的协议，可在其他地方35。 </li><li>清洁的8"硅晶片在100瓦用氧等离子体处理5分钟。 </li><li>倒入足够的负性光刻胶（SU-82100效果很好）到晶片，使抵抗形式的2.5厘米直径的一团。使用旋涂器，旋晶片在500rpm以100rpm /秒斜坡并保持10秒。然后上升到1600 rpm的转速300转/秒，保持30秒。注:速度和斜坡需要为每个实验室条件进行优化，以得到150μm的抗蚀剂厚度。 </li><li>软烘烤在65℃下7分钟的晶片，斜坡上升至95℃以360℃/小时，保持45分钟。慢慢冷却到室温在热板上。由铬光掩模下曝光于365nm UV光压印容器图案上涂覆的晶片，用曝光三倍制造商的建议（350毫焦/厘米2的大约150微米的厚度的SU-8）的。 注意:由于SU-8是一种负性抗蚀剂中，曝光区域（除了图案设计的所有内容）将变得不溶于在步骤1.1.7显影剂。 </li><li>硬烘烤65露出的晶片℃进行5分钟，并上升到95℃与360℃/小时，保持25分钟，然后允许其缓慢冷却到室温在热板上。 </li><li>浸入SU-8开发晶圆10 - 15分钟洗去未曝光的抵抗，然后用异丙醇清洁和干燥压缩氮气流中。检查在光学显微镜下的图案，以确保对SU-8抗蚀剂被很好地结合到晶片（寻找不必要剥离和气泡）。 </li><li>测量使用根据制造商的说明36轮廓曲线的图案特征的厚度。在测量过程中获取，避免对于网络功能（ 即，入口和出口导管），为基于接触轮廓可能损害在晶片上的图形化功能部件的结构完整性所必需的结构的区域。另外，使用非接触方式（ 即光学轮廓仪）完全避免这个问题。 </li></ol></li><li> 通用电器图案和平板模具neration胶原成型 注:化学通风柜内拉手硅烷。 <ol><li>放置晶片用100μl三氯（3,3,3-三氟丙基）硅烷的干燥器2小时到silanize表面。 </li><li>硅烷化转移晶片制成120×120平方毫米的培养皿。倒混合并脱气的PDMS弹性体和固化剂（10:1重量/重量比）在晶片上，以实现4 - 第6毫米的厚度。另外倒入PDMS到一个单独的120×120平方毫米的培养皿中生成平模具无模式。固化在65℃下2小时。 </li><li>从烤箱中取出，并允许PDMS冷却到室温。使用手术刀小心切开围绕的SU-8的正方形和慢慢剥离从晶片的PDMS模具。修剪边缘至30mm×30毫米。适用于平面模具，剪治愈PDMS没有压印花纹小方块约为40mm×40毫米。 </li></ol></li></ol> 2.房屋设备<ol><li> Fabricat顶离子和底部外壳件<ol><li>制造使用的聚（甲基丙烯酸甲酯）（PMMA）的容器壳体。为了制作，从一个标准的机加工车间与计算机数控（CNC）铣削功能订购的部分。参见图 1的顶部和底部件的示意图。 </li><li>设计顶壳件（ 图1D），以包括20毫米×20mm的孔上的设备的为1毫米的深度在位于四角的装置的顶部下侧，二胶原喷射口（直径4 mm直径）方形的孔，两个出入口油藏6毫米直径，并且在该装置的四角四个螺孔（直径为3毫米）。 注意:附加孔可以对片进行处理的目的的周边钻孔。 </li><li>设计底部外壳件（ 图1E），包括在中间的方孔（尺寸15毫米×15毫米）与周围25毫米×25mm的架具有0.25毫米的深度。钻4个螺丝孔与4-40线程。确保在底部的螺丝孔和顶部的作品将在今后的组装步骤一起对齐。 </li></ol></li></ol> 3.微血管器件制造<ol><li> 材料的制备 <ol><li>洗和高压灭菌两套镊子，不锈钢刮刀，一个小的扁平螺丝刀，不锈钢螺丝和几个不锈钢定位销。为容器的制造中，也进行高压灭菌微图案的PDMS模具，PDMS平坦正方形，玻璃盖玻片（22×22 MM）和棉片。 </li><li>消毒在漂白剂的PMMA壳件，至少1小时，然后在组织培养橱蒸压水冲洗两次，并允许在无菌组织培养皿空气干燥（150毫米×25毫米）。 </li></ol></li><li> 无菌聚乙烯亚胺-戊二醛（PEI - GA）涂层 <ol><li>对待消毒干PMMA片的外侧井与等离子体约1分钟。添加1％聚乙烯亚胺（PEI）到内孔中（20毫米见方以及在顶部和底部25mm的正方形货架）聚甲基丙烯酸甲酯片10分钟的。用无菌H 2Ø冲洗并在组织培养罩干燥。 注意:需要的等离子体处理的时间是可变的取决于所使用的设备上 - PEI的应该容易传播表示的亲水性表面。如果不是这样，附加的等离子体处理可能是必要的。 </li><li>应用0.1％戊二醛（GA）到PEI处理的聚甲基丙烯酸甲酯片的表面30分钟。用无菌水冲洗两次，晾干。 注:在未来的步骤，胶原蛋白会通过胶原交联戊二醛附着在涂层表面。这有助于在未来的组装步骤和整个装置文化时期固定在设备内发生凝胶。 </li></ol></li><li> 胶原凝胶制备 <ol><li>制造使用0.6％的船只 - 1％的胶原蛋白解决方案。为了使对容器制造0.75％的胶原蛋白，拌I型胶原蛋白原液（1.5％ -从大鼠分离尾部37）用氢氧化钠（NaOH），10倍的媒体补充（M199），和细胞培养介质。置于冰上所有的解决方案。 </li><li>通过下面的等式，其中Vƒinal是需要凝胶的最终体积确定胶原蛋白的量（通常为1毫升每个设备胶原的就足够了），V 股 ​​票胶原是所需库存胶原蛋白的量， 碳库胶原是浓度股票的胶原蛋白，和Cƒinal胶原是在容器中的胶原蛋白的所需的浓度。 <img alt="式（1）" src="/files/ftp_upload/54457/54457eq1.jpg" /></li><li>用1ml注射器向库存胶原的适当体积转移至新的30毫升锥形管中。确定中和氢氧化钠（V 氢氧化钠 ）的体积，M199 10X媒体的补充（V 10 X）和细胞培养基（Ⅴ1Ⅹ）如下和在15ml锥形管中分别混合: <img alt="公式（2）" src="/files/ftp_upload/54457/54457eq2.jpg" /></li><li>添加中和试剂（V 10 X，V 氢氧化钠 ，V 1 x）的等分胶原蛋白的混合物中，并用小刮铲，直至获得均质凝胶轻轻混合溶液。避免搅拌下慢慢，轻轻地引入气泡。 </li><li>为了将细胞进入基质在所希望的浓度（ 例如，0.5年- 2000万元细胞/ ml），将细胞加入到胶原溶液其与中和试剂混合后，并继续搅拌直至细胞均匀分布。 </li><li>当加入细胞，调整细胞培养基的体积，以容纳附加的体积，如通过下面的等式，其中V 细胞来确定&lt;/子&gt;是细胞悬浮液所要求的体积，Cƒinal细胞密度是在容器的细胞的期望的密度，C 悬浮细胞密度是细胞在原液的密度。 <img alt="公式3" src="/files/ftp_upload/54457/54457eq3.jpg" /></li></ol></li><li> 胶原蛋白注射-顶件 <ol><li>放在100毫米×20毫米的菜的微图案PDMS模。等离子体清洁的PDMS模具1分钟。 </li><li>对齐的PDMS模具的顶部的顶部的PMMA片，使得模具上的正方形储层直属入口和出口储（ 见图1D -虚线）。将不锈钢定位销到入口和出口的顶部PMMA片水库孔，以保持与水库花纹清晰访问。 </li><li>用1ml注射器抽取〜0.5 - 0.6毫升胶原。确保没有气泡残留在注射器中。 </li><li>按下倒升ightly与顶部壳体上镊子以确保顶部件和PDMS模具之间齐平的接触。通过在顶部的PMMA片的注射口注射胶原慢。检查胶原20mm的×20mm的领域上面的图案填充，不会泄漏出来。 </li><li>关闭盘不拥挤的定位销，并允许在37℃培养箱胶凝30分钟。 </li></ol></li><li> 胶原蛋白注射-底件 <ol><li>放置一个22×22毫米的玻璃盖玻片在底部件的中心。用1ml注射器均匀分配〜0.25毫升胶原到玻璃载玻片。轻轻降低PDMS平面方形过的胶原蛋白，以确保与PDMS是针对PMMA平面和没有捕获的气泡。允许在37℃胶凝30分钟。 </li></ol></li><li> 设备装配 <ol><li>凝胶化后，在底部件补充足够的PBS周围的平坦PDMS片包围的PDMS（约1ml）。使用镊子取出任何电子邮件xcess胶原周围的边缘，并慢慢剥去PDMS片。添加更多的PBS的胶原蛋白来维持水化顶部。 </li><li>为了准备顶部件，用镊子拿起顶部PMMA一块，将其翻转过来，以使PDMS模具之上。添加PBS几滴到界面，然后迅速和坚定地取下顶部P​​MMA片PDMS模。 </li><li>轻轻地取出由PMMA壳的另一侧用另一个镊子不锈钢定位销。添加PBS的几个滴在微图案的胶原蛋白。翻转PMMA顶件以上，使胶原朝下，并把4个螺钉成角孔。 </li><li>轻轻躺在底部件的顶部的顶板，对准在底部件的拐角处的孔的螺钉。不要让两件相互滑动。用螺丝刀拧紧用温柔的触摸螺丝。不要拧得过紧。 </li><li>吸出PBS周边PMMA表面并放置一个小圆周率根据该装置的一个边缘棉花ECE。使用移液管，从容器除去任何的PBS并用细胞培养基代替。允许设备在37℃培养箱在一起凝胶至少1小时。 </li></ol></li><li> 细胞种植 <ol><li>培养人脐静脉内皮细胞（HUVECs）的内皮生长培养基在37℃（CO 2，O 2）至汇合38。只要有可能，通道4和7之间使用。 </li><li>通过用6ml PBS中洗涤培养烧瓶中，然后在37℃加入0.05％胰蛋白酶2毫升以解离细胞Trypsinize内皮细胞。离开细胞胰蛋白酶直到大部分粘着斑具有离并将细胞四舍五入（这通常需要约2 - 3分钟）。停止加入4毫升含有牛胎儿血清（FBS）的内皮生长培养基的胰蛋白酶反应。洗与媒体几次烧瓶中以确保将细胞从烧瓶中分离，并收集在锥形管中。 </li><li>算使用血球细胞中并在每毫升1000万个单元的密度重悬。从入口和出口储除去所有的细胞培养基。用200μl凝胶加载提示，加10微升细胞悬浮液到入口储的中心。细胞应该开始流入紧接网络和外套网络。 </li><li>同样加入200μl媒体两者水库和让细胞附着和网络内至少1小时传播。 </li></ol></li><li> 设备文化 <ol><li>培养通过添加200微升的细胞培养基向入口储和50μl到出口储槽与通过网络重力驱动流的装置。随着文化的这种方法，更换介质每12小时，以保持血管内皮的可行性。 注意:如果连续流动的培养条件是所希望的（保持恒定的剪切应力，在出口处， 等等收集介质）一SYR英格泵可以用来代替重力驱动的文化。 </li><li>允许种子内皮细胞至少24小时的连续申请前流附加和稳定。此前连续流设置，保持与重力驱动流动条件的设备。 注:应用流媒体条件与重力驱动的文化差异。增加了一个等离子体膨胀，如葡聚糖，向媒体稳定工程改造微血管和持续灌注39期间防止血管塌陷。 </li><li>为了让媒体连续流设置，在内皮细胞生长介质溶解3.5％葡聚糖（70 kDa的）最佳船文化。使用无菌技术，填补10毫升注射器与内皮细胞生长介质3.5％右旋糖酐。 </li><li>通过使用烙铁熔化在培养皿的侧壁孔制备用于流的无菌培养皿中。 </li><li>附加12"硅管（1/32"ID），以鲁尔锁连接（女，1/16"ID）和一个1 /16"直管与管连接器与一个1"在另一端管段。插入一个1/4"20G钝针（从针毂分离的）至1的自由端"段。还准备附连到管与管90°弯头连接器接头（将被装配到壳体入口），在随后的步骤中，较长的管将通过准备菜螺纹并安装于3管道的3"段通过20G的针"段。出口管路应反映入口管，具有一个自由端，而不是一个路厄锁定连接。高压灭菌，使用前所有段。 </li><li>通过准备好的菜孔螺纹高压入口和出口管路。连接3"的段到内20G针。附加在Luer锁耦合到填充介质注射器，并通过使用注射器泵设定在一个较高的流速管灌注介质。确保有在管道或接头无气泡，因为这些将阻碍流动到容器中。 </li><li>插入的Connector关节入壳体入口。设定注射器泵以所需的流速和开始灌注。 注意:此可根据容器的几何形状和所施加的剪切应力的实验条件进行调整。对于100微米直径的通道，3微升的流速/分钟效果很好。 </li><li>如果需要，收集灌流液与插入含有500微升媒体无菌5毫升聚苯乙烯圆底管准备出口管道。 注意:介质少量加入到收集管，以防止气泡的形成，可以阻止流动。 </li><li>取决于流速，注射器可能需要后24至36小时，再填充。这是通过从壳体入口移除连接器，取代了注射器，并且允许媒体以高流速几分钟灌注完成。这个序列可确保气泡不会被引入到管道。重置泵到所需的流速为培养，插入连接器插入壳体入口，并返回到培养箱中。 </li></ol></li></ol> 4.设备分析<ol><li> 渗透性分析 <ol><li>使用40 kDa的FITC-葡聚糖形象化原位血管的渗透性。广场上共聚焦显微镜血管住房和集中在容器面。添加5μM的40 kDa的FITC-葡聚糖在4X放大，25毫秒的曝光时间在入口和图像，并以每秒1帧为10分钟。 </li><li>分析与分析代码图像系列基于由Zheng 等发表的模型来估计葡聚糖的渗透性穿过血管壁进入胶原（微米/秒）。人 31。 </li></ol></li><li> 免疫组化和共焦成像分析 <ol><li>要修复的船只，由灌注PBS冲洗三次（每次洗涤20分钟）灌注用3.7％的甲醛通过20分钟，并洗涤所述入口。块的非特异性抗体具有2％牛血清白蛋白（BSA）和0.5％的Triton X-100 PE结合通过网络1小时rfused。在4℃下过夜灌注初级抗体溶液并用PBS洗涤三次。灌注次级抗体溶液为1 - 2小时，并以相同的方式，用PBS再次洗涤。 </li><li>使用共聚焦显微镜的1微米的z步骤采取原位微血管免疫荧光图像。图像在4X，10X，或20X的放大倍数的微血管。 注意:4X的倍率将提供全球网络结构的信息，而10X和20X的放大图像将提供蜂窝的细节，如伸长率，结形成， 等等 。 </li><li>分析使用ImageJ以Z预测（图片/栈/ Z-项目），横截面（图片/栈/正交视图）和三维重建（图片/栈/ 3D项目）图像堆栈。 </li></ol></li><li> 扫描电子显微影像 <ol><li>对于电子显微镜分析， 修复原位灌注半强度Karnovsky的溶胶ution（在0.2M的二甲胂缓冲液2％多聚甲醛/ 2.5％戊二醛）过夜。拆卸船只，小心地将两件分开。修剪边，并在几天固定液充分浸泡。 </li><li>沉浸在25％的戊二醛在容器的厚顶部部分20分钟，并用PBS冲洗三次（每次2分钟）。脱水在50％，70％，85％（每次2分钟），并在100％乙醇（每次5分钟）洗涤两次串行乙醇洗涤。 </li><li>准备用于与按照制造商的协议40进一步脱水临界点干燥分析样品。溅射涂覆容器用金 - 钯（7纳米）和扫描电子显微镜下分析用5千伏，光斑尺寸3的加速电压。 </li></ol></li></ol>

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作者什么都没有透露。

改造的微血管是一个体外模型，其中诸如鲁米几何形状，流体动力，和多细胞相互作用生理特性是存在和可调。这种类型的平台的是，它提供模拟和在各种情况下，其中在体外培养条件可以匹配，有关的微环境的研究内皮行为的能力强大。例如，驾驶内皮过程，如血管生成的机制，已知在不同器官和在不同的病理状态，例如健康和疾病44不同发生。出于这个原因，平面内皮细...

各器官微血管帮助定义组织微环境，保持组织的平衡和调节炎症反应，透气性，血栓形成，纤溶1,2。的微血管内皮，尤其是血流和周围组织并因此之间的界面起着响应于刺激如流体动力和循环细胞因子和激素3调制血管和器官功能起关键作用- 5。理解内皮，血液之间的详细的相互作用，与周围组织的微环境是血管生物学和疾病进展的研究很重要。然而，在研究这些相互作用的进展已经阻碍通过体外工具不体内微血管结构概括和功能6,7-限制。其结果是，外地和治疗的进步主要依赖昂贵和耗时消费的动物模型，往往不能转化为人类的8成功- 10。而在体内模型中的疾病机制和血管功能的研究非常宝贵的，它们是复杂的，且往往缺乏个体细胞，生化的精确控制，和生物物理提示。 血管遍布全身拥有与扩张毛细血管床结合了成熟的分层结构，提供优化的灌注和养分运输同时11。最初，血管的形式作为早期发育过程中12,13整理到一个层次支网络原始丛。尽管许多参与这些过程的信号的充分理解14 - 16日 ，它的血管形成图案是如何这样确定的15仍然难以捉摸。反过来， 在体外扼要这个过程中为工程师举办的血管网络具有蜂Ñ ​​困难的。许多现有体外平台建模脉管，如二维内皮细胞培养物，缺乏如多细胞接近度，三维管腔几何形状，流量，和细胞外基质的重要特征。在三维水凝胶管形成测定（胶原或纤维蛋白）17 - 19或入侵检测20,21已被用于研究在3D和它们之间的相互作用内皮功能与其他血管17,22或组织的细胞类型23。然而，组装在这些测定流明缺乏互连性，血流动力学流动，以及适当的灌注。此外，对于在这些管形成测定24血管退化的倾向防止长期培养和成熟这限制了可以进行功能性研究的程度。因此，存在一个新兴需要工程师体外微血管网络，可以适当地进行建模连接的平台内皮特性，并能够长期培养的。 各种血管的工程技术已经出现多年来为医疗应用，以取代或患有血管疾病旁路血管的影响。如聚乙烯对苯二甲酸酯（PET），和聚四氟乙烯（ePTFE）由合成材料制成的大直径容器已经与长期通畅（平均95％的通畅超过5年）25相当的治疗成功。虽然小直径合成移植物（&lt;6毫米）通常面临的并发症，如内膜增生和血栓形成26 - 28日 ，组织工程与生物材料制成的小直径移植取得了显著的进展29,30。尽管有这样的进步，在微尺度工程船仍然是一个挑战。到微血管充分模拟，有必要产生具有萨夫复杂网络模式够的机械强度，以保持通畅，并用基质组合物，其允许为实质细胞和细胞重塑既养分渗透。 该协议提出了一种新的人工血管perfusable网络模拟体内的天然具有可调可控的微环境设置31 - 34。所描述的方法生成的直径为100微米量级上的工程微血管。工程微血管由通过嵌入软I型胶原蛋白凝胶中的微流体通道灌流内皮细胞制造。该系统具有生成具有开放的管腔结构图案网络，复制的多细胞相互作用，调节细胞外基质成分，并应用生理学相关的血流动力学部队的能力。

<table><tbody><tr><td>&lt;strong&gt;Wafer Fabrication&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>AutoGlow Plasma System</td><td>AutoGlow</td><td></td><td></td></tr><tr><td>Headway Spin Coater</td><td>Headway Research, Inc&amp;nbsp;</td><td>PWM32 Spin Coater&amp;nbsp;</td><td></td></tr><tr><td>ABM Contact Aligner</td><td>AB-M</td><td></td><td></td></tr><tr><td>Alpha Step Profilometer</td><td>Tencor</td><td>Alpha Step 200</td><td></td></tr><tr><td>SU-8 Developer</td><td>Microchem</td><td>Y020100</td><td></td></tr><tr><td>SU-8 Resist</td><td>Microchem</td><td>SU-8 2000</td><td></td></tr><tr><td>8&quot; silicon wafer</td><td>Wafer World Inc.</td><td></td><td></td></tr><tr><td>Tabletop Micro Pattern Generator</td><td>Heidelberg Instruments</td><td>&amp;mu;PG 101</td><td>For generation of photomask</td></tr><tr><td>Hot plate</td><td>VWR</td><td>97042-646</td><td></td></tr><tr><td>Ispropyl alcohol</td><td>Avantor Performance Materials</td><td>9088</td><td></td></tr><tr><td>Petri dishes (120 x 120 mm, square)</td><td>Sigma-Aldrich</td><td>Z617679</td><td></td></tr><tr><td>Trichloro(3,3,3-trifluoropropyl)silane</td><td>Sigma-Aldrich</td><td>MKBG3805V</td><td></td></tr><tr><td>Polydimethylsiloxane (PDMS) elastomer base and curing agent</td><td>Dow Corning</td><td>Sylgard 184</td><td>Mixed at 10:1 (w/w)</td></tr><tr><td>Vacuum desiccator</td><td>Sigma-Aldrich</td><td>Z119024-1EA</td><td></td></tr><tr><td>Oven</td><td>VWR</td><td>9120976</td><td></td></tr><tr><td>&lt;strong&gt;Device Fabrication and Culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>poly(methyl methacrylate) (PMMA)</td><td>Plexiglas</td><td></td><td></td></tr><tr><td>Corona Treater</td><td>Electro-Technic Products, Inc.</td><td>BD-20</td><td>Handheld device for plasma treatment of PMMA devices and PDMS molds</td></tr><tr><td>Soldering Iron</td><td>Weller&amp;nbsp;</td><td>WTCPS</td><td></td></tr><tr><td>Stainless Steel Truss Head Slotted Machine Screw</td><td>McMaster-Carr&amp;nbsp;</td><td>91785A096</td><td></td></tr><tr><td>Stainless steel dowel pins</td><td>McMaster-Carr&amp;nbsp;</td><td>93600A060</td><td></td></tr><tr><td>Tweezers&amp;nbsp;</td><td>Miltex</td><td>24-572</td><td>Any similar tweezers may be used</td></tr><tr><td>Spatula (Micro Spoon)</td><td>Electron Microscopy Services</td><td>62410-01</td><td></td></tr><tr><td>Screw driver</td><td></td><td></td><td>Any flat head screwdriver may be used, autoclaved</td></tr><tr><td>Glass coverslips (22 x 22 mm)</td><td>Fisher Scientific</td><td>12-542B</td><td></td></tr><tr><td>Bleach</td><td>Clorox</td><td>4460030966</td><td></td></tr><tr><td>Petri dishes (150 x 25 mm)</td><td>Corning</td><td>430599</td><td></td></tr><tr><td>Petri dishes (100 x 20 mm)</td><td>Corning</td><td>2909</td><td></td></tr><tr><td>Cotton, cut into 1 cm x 3 cm pieces</td><td></td><td></td><td>Autoclaved</td></tr><tr><td>Polyethyleneimine (PEI)</td><td>Sigma-Aldrich</td><td>P3143</td><td>Dilute to 1% in cell culture grade water</td></tr><tr><td>Glutaraldehyde</td><td>Sigma-Aldrich</td><td>G6257</td><td>Dilute to 0.1% in cell culture grade water</td></tr><tr><td>Sterile H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>Autoclaved DI H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Type I collagen, dissolved in 0.1% acetic acid</td><td></td><td></td><td>Isolated from rat tails as described in Rajan &lt;i&gt;et. al.&lt;/i&gt; 2006 (ref #37)</td></tr><tr><td>1 ml syringe</td><td>BD</td><td>309659</td><td></td></tr><tr><td>10 ml syringe</td><td>BD</td><td>309604</td><td></td></tr><tr><td>15 ml conical tubes</td><td>Corning</td><td>352097</td><td></td></tr><tr><td>30 ml conical tubes</td><td>Corning</td><td>352098</td><td></td></tr><tr><td>M199 10x Media&amp;nbsp;</td><td>Life Technologies</td><td>11825-015</td><td></td></tr><tr><td>1 N NaOH (sterile)</td><td>Sigma-Aldrich</td><td>415413</td><td>Dilute to 1 N in cell culture grade water</td></tr><tr><td>HUVECs</td><td>Lonza</td><td></td><td></td></tr><tr><td>Endothelial growth media</td><td>Lonza</td><td>CC-3124</td><td></td></tr><tr><td>Trypsin</td><td>Corning</td><td>25-052-CI</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Thermofisher Scientific</td><td>10082147</td><td></td></tr><tr><td>Dextran from Leuconostoc spp. (70&amp;nbsp;kDa)</td><td>Sigma-Aldrich</td><td>31390</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>Hemocytometer</td><td>Hausser Scientific Co.</td><td>3200</td><td></td></tr><tr><td>Gel loading tips</td><td>VWR</td><td>37001-152</td><td></td></tr><tr><td>18 G Blunt Fill Needle</td><td>BD&amp;nbsp;</td><td>305180</td><td></td></tr><tr><td>20 G Stainless Steel Dispensing Needle</td><td>McMaster-Carr</td><td>75165A123</td><td></td></tr><tr><td>Tygon 1/32&amp;rdquo; ID, 3/32&quot; OD Silicon Tubing</td><td>Cole-Parmer</td><td>EW-95702-00</td><td></td></tr><tr><td>1/16&quot; Tube-to-tube Coupling</td><td>McMaster-Carr</td><td>5116K165</td><td></td></tr><tr><td>90&amp;deg; Elbow Connectors, Tube-to-Tube</td><td>McMaster-Carr</td><td>5121K901</td><td></td></tr><tr><td>Luer Lock Coupling (Female, 1/16&quot; ID)</td><td>McMaster-Carr</td><td>51525K211</td><td></td></tr><tr><td>Plastic Forceps, with Jaw Grips</td><td>Electron Microscopy Services</td><td>72971</td><td></td></tr><tr><td>Dual Syringe Pump</td><td>Harvard Apparatus</td><td>70-4505</td><td></td></tr><tr><td>5 ml Polystyrene Round-bottom tube</td><td>Fisher Scientific</td><td>14-959-2A</td><td></td></tr><tr><td>&lt;strong&gt;Device Analysis&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Formaldehyde</td><td>Sigma-Aldrich</td><td>F8775</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A8806-5G</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T-9284</td><td></td></tr><tr><td>Rabbit anti-hCD31</td><td>Abcam</td><td>ab32457</td><td>1:25 working dilution</td></tr><tr><td>FITC conjugated anti-von Willebrand Factor antibody</td><td>Abcam</td><td>ab8822</td><td>1:100 working dilution</td></tr><tr><td>Goat anti-rabbit 568 secondary antibody</td><td>Thermofisher Scientific</td><td>A-11011</td><td>1:100 working dilution</td></tr><tr><td>Hoescht</td><td>Thermofisher Scientific</td><td>H1399</td><td>Resuspended in DMSO</td></tr><tr><td>Sodium cacodylate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C0250</td><td>To make 0.2 M cacodylate buffer</td></tr><tr><td>Ethanol</td><td>VWR International</td><td>BDH1164-4LP</td><td></td></tr><tr><td>40 kDa FITC-conjugated Dextran</td><td>Sigma-Aldrich</td><td>FD40S&amp;nbsp;</td><td></td></tr><tr><td>&lt;strong&gt;Additional Culture Reagents&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CHIR-99021</td><td>Selleck Chem</td><td>S2924</td><td>Small molecule GSK-3 inhibitor</td></tr><tr><td>Human recombinant VEGF</td><td>Peprotech</td><td>100-20</td><td></td></tr><tr><td>Human recombinant bFGF</td><td>Peprotech</td><td>AF-100-18B</td><td></td></tr></tbody></table>

micropatterning and assembly of 3d microvessels

体外平台研究血管内皮细胞和血管生物学很大程度上限于2D内皮细胞培养物，与聚合物或基于玻璃基板，和基于水凝胶的管形成测定流动腔室。这些测定，而信息量大，不概括腔的几何形状，正确的细胞外基质，和多细胞的接近，这在调节血管功能中发挥关键作用。该原稿描述了一种注射成型方法，以产生直径为100μm量级的工程化血管。微血管被嵌入的本地I型胶原凝胶内的微流体通道播种内皮细胞制造。通过通道形成之前胶原基质内掺入实质细胞，组织特异性微环境进行建模和分析。流体力学性质和介质组合物的附加的调制允许对所需的微环境内复杂的血管功能的控制。这个平台允许血管周围细胞募集，血内皮细胞相互作用，流动响应，并组织微血管相互作用的研究。工程改造微血管提供来隔离从血管利基的单个组件的影响的能力，并精确地控制其化学，机械和生物性质，研究血管生物学两个健康和疾病。

该工程船平台创建嵌入式天然I型胶原基质内的微血管功能，并允许进行体外细胞，生物物理和生物化学环境的严格控制。为了制造工程改造的微血管，人脐静脉内皮细胞（HUVECs）通过胶原包埋微流体网络，其中它们连接，以形成一个专利腔和汇合内皮灌注。如在图1A-C中所示，容器的几何形状可以被专门设计用于回答有关的流速，扭曲的问题，和分支角...

Watch this Scientific Journal Video about Micropatterning and Assembly of 3D Microvessels at JoVE.com

Micropatterning and Assembly of 3D Microvessels

该原稿提出了一种注射模制方法来设计该概括内皮生理特性微血管。基于微流体过程创建具有可调整的条件，如流量，细胞组成，几何形状和生化梯度专利3D血管网络。制造过程和潜在的应用例子中描述。

缩微和3D微血管大会

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

micropatterning-and-assembly-of-3d-microvessels

Research

JoVE Journal

Bioengineering

11.7K 次观看.  University of Washington. 该原稿提出了一种注射模制方法来设计该概括内皮生理特性微血管。基于微流体过程创建具有可调整的条件，如流量，细胞组成，几何形状和生化梯度专利3D血管网络。制造过程和潜在的应用例子中描述。 

视频: Micropatterning and Assembly of 3D Microvessels

Process of Making Three-dimensional Microstructures using Vaporization of a Sacrificial Component

Vascular structures in natural systems are able to provide high mass transport through high surface areas and optimized structure. Few synthetic material fabrication techniques are able to mimic the complexity of these structures while maintaining scalability. The Vaporization of a Sacrificial Component (VaSC) process is able to do so. This process uses sacrificial fibers as a template to form hollow, cylindrical microchannels embedded within a matrix. Tin (II) oxalate (SnOx) is embedded within poly(lactic) acid (PLA) fibers which facilitates the use of this process. The SnOx catalyzes the depolymerization of the PLA fibers at lower temperatures. The lactic acid monomers are gaseous at these temperatures and can be removed from the embedded matrix at temperatures that do not damage the matrix. Here we show a method for aligning these fibers using micromachined plates and a tensioning device to create complex patterns of three-dimensionally arrayed microchannels. The process allows the exploration of virtually any arrangement of fiber topologies and structures.

The Vaporization of a Sacrificial Component (VaSC) process is used to fabricate microvascular structures. This procedure uses sacrificial poly(lactic) acid fibers to form hollow microchannels with precise 3D geometric positioning provided by laser micromachined guide plates.

制作三维微结构，用牺牲部分汽化过程

Vascular structures in natural systems are able to provide high mass transport through high surface areas and optimized structure. Few synthetic material ...

科研

工程学

A Microfluidic Device with Groove Patterns for Studying Cellular Behavior

We describe a microfluidic device with microgrooved patterns for studying cellular behavior. This microfluidic platform consists of a top fluidic channel and a bottom microgrooved substrate. To fabricate the microgrooved channels, a top poly(dimethylsiloxane) (PDMS) mold containing the impression of the microfluidic channels was aligned and bonded to a microgrooved substrate. Using this device, mouse fibroblast cells were immobilized and patterned within microgrooved substrates (25, 50, 75, and 100 &mu;m wide). To study apoptosis in a microfluidic device, media containing hydrogen peroxide, Annexin V, and propidium iodide was perfused into the fluidic channel for 2 hours. We found that cells exposed to the oxidative stress became apoptotic. These apoptotic cells were confirmed by Annexin V that bound to phosphatidylserine at the outer leaflet of the plasma membrane during the apoptosis process. Using this microfluidic device with microgrooved patterns, the apoptosis process was observed in real-time and analyzed by using an inverted microscope containing an incubation chamber (37&deg;C, 5% CO2). Therefore, this microfluidic device incorporated with microgrooved substrates could be useful for studying the cellular behavior and performing high-throughput drug screening.

We describe a protocol for the fabrication of microfluidic devices that can enable cell capture and culture. In this approach patterned microstructures such as grooves within microfluidic channels are used to create low shear stress regions within which cell can dock.

微流体装置与Groove的模式，为研究细胞行为

We describe a microfluidic device with microgrooved patterns for studying cellular behavior.  This microfluidic platform consists of a top fluidic channel ...

生物学

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

芯片尺寸支架的三维细胞培养的微细

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

Capillary-based Centrifugal Microfluidic Device for Size-controllable Formation of Monodisperse Microdroplets

Here, we demonstrate a simple method for the rapid production of size-controllable, monodisperse, W/O microdroplets using a capillary-based centrifugal microfluidic device. W/O microdroplets have recently been used in powerful methods that enable miniaturized chemical experiments. Therefore, developing a versatile method to yield monodisperse W/O microdroplets is needed. We have developed a method for generating monodisperse W/O microdroplets based on a capillary-based centrifugal axisymmetric co-flowing microfluidic device. We succeeded in controlling the size of microdroplets by adjusting the capillary orifice. Our method requires equipment that is easier-to-use than with other microfluidic techniques, requires only a small volume (0.1-1 &#181;l) of sample solution for encapsulation, and enables the production of hundreds of thousands number of W/O microdroplets per second. We expect this method will assist biological studies that require precious biological samples by conserving the volume of the samples for rapid quantitative analysis biochemical and biological studies.

Here, we demonstrate a simple production method for size-controllable, monodisperse, water-in-oil (W/O) microdroplets using a capillary-based centrifugal microfluidic device. This method requires only a small sample volume and enables high-yield production. We expect this method will be useful for rapid biochemical and cellular analyses.

基于毛细管的离心微流体装置单分散微滴的大小可控的形成

Here, we demonstrate a simple method for the rapid production of size-controllable, monodisperse, W/O microdroplets using a capillary-based centrifugal ...

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

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

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

生物膜的微阵列用刮墨刀型组合方式形成

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

生物工程

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This emerging field engages engineers, chemists, biologists, and physicists to design and assemble basic biological components into complex, functioning systems from the bottom up. Such bottom-up systems could lead to the development of artificial cells for fundamental biological inquiries and innovative therapies1,2. Giant unilamellar vesicles (GUVs) can serve as a model platform for synthetic biology due to their cell-like membrane structure and size. Microfluidic jetting, or microjetting, is a technique that allows for the generation of GUVs with controlled size, membrane composition, transmembrane protein incorporation, and encapsulation3. The basic principle of this method is the use of multiple, high-frequency fluid pulses generated by a piezo-actuated inkjet device to deform a suspended lipid bilayer into a GUV. The process is akin to blowing soap bubbles from a soap film. By varying the composition of the jetted solution, the composition of the encompassing solution, and/or the components included in the bilayer, researchers can apply this technique to create customized vesicles. This paper describes the procedure to generate simple vesicles from a droplet interface bilayer by microjetting.

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

脂质双层囊泡代使用微流控喷射

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

Patterning of Microorganisms and Microparticles through Sequential Capillarity-assisted Assembly

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, including studies of microbial physiology and interactions. At the simplest level, accurate spatial patterning of microorganisms would enable reliable, long-term imaging of large numbers of individual cells and transform the ability to quantitatively study distance-dependent microbe-microbe interactions. More uniquely, coupling accurate spatial patterning and full control over environmental conditions, as offered by microfluidic technology, would provide a powerful and versatile platform for single-cell studies in microbial ecology. 
This paper presents a microfluidic platform to produce versatile and user-defined patterns of microorganisms within a microfluidic channel, allowing complete optical access for long-term, high-throughput monitoring. This new microfluidic technology is based on capillarity-assisted particle assembly and exploits the capillary forces arising from the controlled motion of an evaporating suspension inside a microfluidic channel to deposit individual microsized objects in an array of traps microfabricated onto a polydimethylsiloxane (PDMS) substrate. Sequential depositions generate the desired spatial layout of single or multiple types of micro-sized objects, dictated solely by the geometry of the traps and the filling sequence. 
The platform has been calibrated using colloidal particles of different dimensions and materials: it has proven to be a powerful tool to generate diverse colloidal patterns and perform surface functionalization of trapped particles. Furthermore, the platform was tested on microbial cells, using Escherichia coli cells as a model bacterium. Thousands of individual cells were patterned on the surface, and their growth was monitored over time. In this platform, the coupling of single-cell deposition and microfluidic technology allows both geometric patterning of microorganisms and precise control of environmental conditions. It thus opens a window into the physiology of single microbes and the ecology of microbe-microbe interactions, as shown by preliminary experiments.

We present a technology that uses capillarity-assisted assembly in a microfluidic platform to pattern micro-sized objects suspended in a liquid, such as bacteria and colloids, into prescribed arrays on a polydimethylsiloxane substrate.

通过顺序毛细管辅助组装对微生物和微粒进行图案化

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, ...

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ- and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

影像引导下，血管源性微流控网络的基于激光的研制

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

A Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

图案蛋白质和细胞的通用的方法

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...

Control of Cell Geometry through Infrared Laser Assisted Micropatterning

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular compartments while circumventing complications arising from natural cell-to-cell variations. To standardize cell shape, cells are either confined in 3D molds or controlled for adhesive geometry through adhesive islands. However, traditional micropatterning techniques based on photolithography and deep UV etching heavily depend on clean rooms or specialized equipment. Here we present an infrared laser assisted micropatterning technique (microphotopatterning) modified from Doyle et al. that can be conveniently set up with commercially available imaging systems. In this protocol, we use a Nikon A1R MP+ imaging system to generate micropatterns with micron precision through an infrared (IR) laser that ablates preset regions on poly-vinyl alcohol coated coverslips. We employ a custom script to enable automated pattern fabrication with high efficiency and accuracy in systems not equipped with a hardware autofocus. We show that this IR laser assisted micropatterning (microphotopatterning) protocol results in defined patterns to which cells attach exclusively and take on the desired shape. Furthermore, data from a large number of cells can be averaged due to the standardization of cell shape. Patterns generated with this protocol, combined with high resolution imaging and quantitative analysis, can be used for relatively high throughput screens to identify molecular players mediating the link between form and function.

The protocol presented here enables automated fabrication of micropatterns that standardizes cell shape to study cytoskeletal structures within mammalian cells. This user-friendly technique can be set up with commercially available imaging systems and does not require specialized equipment inaccessible to standard cell biology laboratories.

通过红外激光辅助微模式控制细胞几何

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular ...