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。

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			<td height="21" style="height:21px;width:459px;">Wafer Fabrication</td>
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			<td style="width:140px;"></td>
			<td style="width:540px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AutoGlow Plasma System</td>
			<td>AutoGlow</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Headway Spin Coater</td>
			<td>Headway Research, Inc&#160;</td>
			<td colspan="2">PWM32 Spin Coater&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ABM Contact Aligner</td>
			<td>AB-M</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alpha Step Profilometer</td>
			<td>Tencor</td>
			<td>Alpha Step 200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SU-8 Developer</td>
			<td>Microchem</td>
			<td>Y020100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SU-8 Resist</td>
			<td>Microchem</td>
			<td>SU-8 2000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">8&#34; silicon wafer</td>
			<td>Wafer World Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tabletop Micro Pattern Generator</td>
			<td>Heidelberg Instruments</td>
			<td>&#956;PG 101</td>
			<td>For generation of photomask</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hot plate</td>
			<td>VWR</td>
			<td>97042-646</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ispropyl alcohol</td>
			<td>Avantor Performance Materials</td>
			<td>9088</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri dishes (120 x 120 mm, square)</td>
			<td>Sigma-Aldrich</td>
			<td>Z617679</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trichloro(3,3,3-trifluoropropyl)silane</td>
			<td>Sigma-Aldrich</td>
			<td>MKBG3805V</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">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 height="17">
			<td height="17" style="height:17px;">Vacuum desiccator</td>
			<td>Sigma-Aldrich</td>
			<td>Z119024-1EA</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Oven</td>
			<td>VWR</td>
			<td>9120976</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Device Fabrication and Culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">poly(methyl methacrylate) (PMMA)</td>
			<td>Plexiglas</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">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 height="20">
			<td height="20" style="height:20px;">Soldering Iron</td>
			<td>Weller&#160;</td>
			<td>WTCPS</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stainless Steel Truss Head Slotted Machine Screw</td>
			<td>McMaster-Carr&#160;</td>
			<td>91785A096</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stainless steel dowel pins</td>
			<td>McMaster-Carr&#160;</td>
			<td>93600A060</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tweezers&#160;</td>
			<td>Miltex</td>
			<td>24-572</td>
			<td>Any similar tweezers may be used</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Spatula (Micro Spoon)</td>
			<td>Electron Microscopy Services</td>
			<td>62410-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Screw driver</td>
			<td></td>
			<td></td>
			<td>Any flat head screwdriver may be used, autoclaved</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass coverslips (22 x 22 mm)</td>
			<td>Fisher Scientific</td>
			<td>12-542B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bleach</td>
			<td>Clorox</td>
			<td>4460030966</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri dishes (150 X 25mm)</td>
			<td>Corning</td>
			<td>430599</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri dishes (100 X 20 mm)</td>
			<td>Corning</td>
			<td>2909</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cotton, cut into 1 cm x 3 cm pieces</td>
			<td></td>
			<td></td>
			<td>Autoclaved</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polyethyleneimine (PEI)</td>
			<td>Sigma-Aldrich</td>
			<td>P3143</td>
			<td>Dilute to 1% in cell culture grade water</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glutaraldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>G6257</td>
			<td>Dilute to 0.1% in cell culture grade water</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Sterile H2O</td>
			<td></td>
			<td></td>
			<td>Autoclaved DI H2O</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Type I collagen, dissolved in 0.1% acetic acid</td>
			<td></td>
			<td></td>
			<td>Isolated from rat tails as described in Rajan et. al. 2006 (ref #37)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 mL syringe</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10 mL syringe</td>
			<td>BD</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15 mL conical tubes</td>
			<td>Corning</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">30 mL conical tubes</td>
			<td>Corning</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">M199 10X Media&#160;</td>
			<td>Life Technologies</td>
			<td>11825-015</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1N NaOH (sterile)</td>
			<td>Sigma-Aldrich</td>
			<td>415413</td>
			<td>Dilute to 1N in cell culture grade water</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HUVECs&#160;</td>
			<td>Lonza</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Endothelial growth media</td>
			<td>Lonza</td>
			<td>CC-3124</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin</td>
			<td>Corning</td>
			<td>25-052-CI</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal bovine serum (FBS)</td>
			<td>Thermofisher Scientific</td>
			<td>10082147</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dextran from Leuconostoc spp. (70kDa)</td>
			<td>Sigma-Aldrich</td>
			<td>31390</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate Buffered Saline (PBS)</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hemocytometer</td>
			<td>Hausser Scientific Co.</td>
			<td>3200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gel loading tips</td>
			<td>VWR</td>
			<td>37001-152</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">18G Blunt Fill Needle</td>
			<td>BD&#160;</td>
			<td>305180</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">20G Stainless Steel Dispensing Needle</td>
			<td>McMaster-Carr</td>
			<td>75165A123</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tygon 1/32&#8221; ID, 3/32&#34; OD Silicon Tubing</td>
			<td>Cole-Parmer</td>
			<td>EW-95702-00</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1/16&#34; Tube-to-tube Coupling</td>
			<td>McMaster-Carr</td>
			<td>5116K165</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">90&#176; Elbow Connectors, Tube-to-Tube</td>
			<td>McMaster-Carr</td>
			<td>5121K901</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Luer Lock Coupling (Female, 1/16&#34; ID)</td>
			<td>McMaster-Carr</td>
			<td>51525K211</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plastic Forceps, with Jaw Grips</td>
			<td>Electron Microscopy Services</td>
			<td>72971</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dual Syringe Pump</td>
			<td>Harvard Apparatus</td>
			<td>70-4505</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5 mL Polystyrene Round-bottom tube</td>
			<td>Fisher Scientific</td>
			<td>14-959-2A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Device Analysis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>F8775</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A8806-5G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T-9284</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rabbit anti-hCD31</td>
			<td>Abcam</td>
			<td>ab32457</td>
			<td>1:25 working dilution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FITC conjugated anti-von Willebrand Factor antibody</td>
			<td>Abcam</td>
			<td>ab8822</td>
			<td>1:100 working dilution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Goat anti-rabbit 568 secondary antibody</td>
			<td>Thermofisher Scientific</td>
			<td>A-11011</td>
			<td>1:100 working dilution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hoescht</td>
			<td>Thermofisher Scientific</td>
			<td>H1399</td>
			<td>Resuspended in DMSO</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium cacodylate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C0250</td>
			<td>To make 0.2M cacodylate buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol</td>
			<td>VWR International</td>
			<td>BDH1164-4LP</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">40kDa FITC-conjugated Dextran</td>
			<td>Sigma-Aldrich</td>
			<td>FD40S&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Additional Culture Reagents&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CHIR-99021</td>
			<td>Selleck Chem</td>
			<td>S2924</td>
			<td>Small molecule GSK-3 inhibitor</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human recombinant VEGF</td>
			<td>Peprotech</td>
			<td>100-20</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">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

Universidad de Cartagena UDEC

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

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo.

A method to obtain nanofibers and complex nanostructures from single or multiple extracellular matrix proteins is described. This method uses protein-surface interactions to create free-standing protein-based materials with tunable composition and architecture for use in a variety of tissue engineering and biotechnology applications.

细胞外基质蛋白纳米纤维和纳米结构工程采用表面引发大会

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber ...

科研

生物工程

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D胶原凝胶和微通道3D相互作用的研究准备<em&gt;在体内</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

模块化 DNA 设备的自动机器人液体处理组件

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.

A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin.

对细胞的超快磁化阳离子化Magnetoferritin的合成

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

微孔阵列平台中微生物群落发展的组装与跟踪

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioink Precellularization 无源混合装置打印软骨及皮肤组织类似物的应用

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

用 3 d-印刷模具制作细胞播种和组织环自组装用的自定义琼脂糖井

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

Assembly and Characterization of Biomolecular Memristors Consisting of Ion Channel-doped Lipid Membranes

The ability to recreate synaptic functionalities in synthetic circuit elements is essential for neuromorphic computing systems that seek to emulate the cognitive powers of the brain with comparable efficiency and density. To date, silicon-based three-terminal transistors and two-terminal memristors have been widely used in neuromorphic circuits, in large part due to their ability to co-locate information processing and memory. Yet these devices cannot achieve the interconnectivity and complexity of the brain because they are power-hungry, fail to mimic key synaptic functionalities, and suffer from high noise and high switching voltages. To overcome these limitations, we have developed and characterized a biomolecular memristor that mimics the composition, structure, and switching characteristics of biological synapses. Here, we describe the process of assembling and characterizing biomolecular memristors consisting of a 5 nm-thick lipid bilayer formed between lipid-functionalized water droplets in oil and doped with voltage-activated alamethicin peptides. While similar assembly protocols have been used to investigate biophysical properties of droplet-supported lipid membranes and membrane-bound ion channels, this article focuses on key modifications of the droplet interface bilayer method essential for achieving consistent memristor performance. Specifically, we describe the liposome preparation process and the incorporation of alamethicin peptides in lipid bilayer membranes, and the appropriate concentrations of each constituent as well as their impact on the overall response of the memristors. We also detail the characterization process of biomolecular memristors, including measurement and analysis of memristive current-voltage relationships obtained via cyclic voltammetry, as well as short-term plasticity and learning in response to step-wise voltage pulse trains.

Soft, low-power, biomolecular memristors leverage similar composition, structure, and switching mechanisms of bio-synapses. Presented here is a protocol to assemble and characterize biomolecular memristors obtained from insulating lipid bilayers formed between water droplets in oil. The incorporation of voltage-activated alamethicin peptides results in memristive ionic conductance across the membrane.

离子通道掺杂脂质膜组成的生物分子记忆器的组装与表征

The ability to recreate synaptic functionalities in synthetic circuit elements is essential for neuromorphic computing systems that seek to emulate the ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

明胶美沙克里洛醇水凝胶生物油墨3D生物印刷方案

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...

Temperature-Controlled Assembly and Characterization of a Droplet Interface Bilayer

The droplet interface bilayer (DIB) method for assembling lipid bilayers (i.e., DIBs) between lipid-coated aqueous droplets in oil offers key benefits versus other methods: DIBs are stable and often long-lasting, bilayer area can be reversibly tuned, leaflet asymmetry is readily controlled via droplet compositions, and tissue-like networks of bilayers can be obtained by adjoining many droplets. Forming DIBs requires spontaneous assembly of lipids into high density lipid monolayers at the surfaces of the droplets. While this occurs readily at room temperature for common synthetic lipids, a sufficient monolayer or stable bilayer fails to form at similar conditions for lipids with melting points above room temperature, including some cellular lipid extracts. This behavior has likely limited the compositions&#8212;and perhaps the biological relevance&#8212;of DIBs in model membrane studies. To address this problem, an experimental protocol is presented to carefully heat the oil reservoir hosting DIB droplets and characterize the effects of temperature on the lipid membrane. Specifically, this protocol shows how to use a thermally conductive aluminum fixture and resistive heating elements controlled by a feedback loop to prescribe elevated temperatures, which improves monolayer assembly and bilayer formation for a wider set of lipid types. Structural characteristics of the membrane, as well as the thermotropic phase transitions of the lipids comprising the bilayer, are quantified by measuring the changes in electrical capacitance of the DIB. Together, this procedure can aid in evaluating biophysical phenomena in model membranes over various temperatures, including determining an effective melting temperature (TM) for multi-component lipid mixtures. This capability will thus allow for closer replication of natural phase transitions in model membranes and encourage the formation and use of model membranes from a wider swath of membrane constituents, including those that better capture the heterogeneity of their cellular counterparts.

This protocol details the use of a feedback temperature-controlled heating system to promote lipid monolayer assembly and droplet interface bilayer formation for lipids with elevated melting temperatures, and capacitance measurements to characterize temperature-driven changes in the membrane.

温度控制组件和液滴接口比莱耶的特征

The droplet interface bilayer (DIB) method for assembling lipid bilayers (i.e., DIBs) between lipid-coated aqueous droplets in oil offers key benefits ...