This work was supported by grants from the National Institutes of Health, including a microphysiological systems consortium grant from the National Center for Advancing Translational Sciences (UH2TR000503), a Ruth L. Kirschstein National Research Service Award Postdoctoral Fellowship (F32DK098905 for WJM) and pathway to independence award (DK095984 for AB) from the National Institute of Diabetes and Digestive and Kidney Diseases.

1.准备本机可溶性胶原溶液中<ol><li>制备或购买200mg的酸化，可溶的，I型大鼠尾胶原在1-3毫克/毫升使用标准的分离协议，诸如报告Piez 等人 15 </li><li>比例基于改性胶原溶液的所需的最终体积上起始原料的量。大约使25-30毫升甲基化和25-30毫升琥珀酰化的胶原蛋白溶液中，每个在3毫克/毫升，从200毫克的可溶性天然胶原。 </li></ol> 2.胶原蛋白甲基化<ol><li>稀释100毫克天然，酸化（pH 2-3）的胶原溶液的为0.5毫克/毫升用冰冷无菌水的浓度，并保持该溶液在冰上，以防止凝胶化。 </li><li>调节胶原溶液以9-10的PH值用几滴1N NaOH和在RT搅拌30分钟。观察胶原沉淀，使溶液变浑浊。 </l我&gt; <li>降速在3000×g下沉淀的胶原溶液25分钟。一种透明，凝胶状沉淀物应当是在管的底部可见。吸并妥善处理上清液的。 </li><li>重悬沉淀的胶原在200ml甲醇中，用0.1N HCl和使甲基化反应，以发生在室温下搅拌4天。胶原蛋白不会解散，而应该分裂成非常小的可见部分，这将使解决方案浑浊。 </li><li>甲基化后，离心该溶液在3000×g的25分钟以沉淀甲基化胶原。吸和处置酸化甲醇上清液。 </li><li>溶解甲基化胶原在25毫升无菌PBS，得到浓度大约为3毫克/毫升，反复移液，并过滤通过60微米的细胞过滤的溶液。调节溶液的使用1N的NaOH 20μl递增pH至7.3-7.4。 </li><li>评估concentratioN使用商业鼠胶原ELISA试剂盒或羟脯氨酸检测试剂盒的解决方案。稀释至3毫克/毫升，用无菌PBS洗涤。 </li><li>通过将其与一螺丝帽转移到玻璃瓶中，小心地分层3毫升氯仿在瓶底消毒甲基化胶原溶液，使瓶子以设置O / N在4℃，然后在无菌条件下取出并存储顶层，它是甲基化的胶原蛋白。 </li><li>存储1个月内溶液在4℃下使用。 </li></ol> 3.胶原蛋白琥珀酰化<ol><li>稀释其他100毫克天然，酸化（pH 2-3）的胶原溶液的为0.5毫克/毫升用冰冷无菌水的浓度，并保持该溶液在冰上，以防止凝胶化。 </li><li>调节胶原溶液以9-10的PH值用几滴1N NaOH和在RT搅拌30分钟。胶原应沉淀，使溶液变浑浊。 </li><li>溶解SUC 40毫克互联网络信息中心酸酐（每胶原毫克0.4毫克）的10毫升丙酮的（1/20的胶原溶液的体积）。缓慢（在大约0.5 ml的增量）加该混合物的胶原溶液在搅拌下，同时连续监测pH。通过加入1或2滴的1N NaOH作为pH值接近9.0维持大于9.0的pH值。 </li><li>继续在RT下加入所有的琥珀酸酐的丙酮后搅拌120分钟。观察溶液变得清澈如琥珀酰化的胶原蛋白溶解。定期检查pH值，以确保它仍然高于9.0。 </li><li>调节溶液至4.0将pH用1N的盐酸20μl递增。再观察溶液混浊，如琥珀酰化的胶原蛋白沉淀。 </li><li>琥珀酰化后，离心该溶液在3000×g的25分钟以沉淀的琥珀酰化的胶原蛋白。吸并丢弃上清液酸化与未反应的琥珀酸酐。 </li><li>化解琥珀胶原蛋白在25ml无菌PBS，得到约3毫克/ ml的浓度，反复移液，并过滤通过60微米的细胞过滤的溶液。调节溶液的使用1N的NaOH 20μl递增pH至7.3-7.4。 </li><li>评估使用市售的大鼠胶原ELISA试剂盒或羟脯氨酸测定试剂盒的溶液的浓度。稀释至3毫克/毫升，用无菌PBS洗涤。 </li><li>通过将其与一螺丝帽转移到玻璃瓶中，小心地分层3毫升氯仿在瓶底消毒琥珀酰化的胶原蛋白溶液，使瓶子以设置O / N在4℃，然后在无菌条件下取出并存储顶层，这是琥珀酰化的胶原蛋白。 </li><li>存储1个月内溶液在4℃下使用。 </li></ol> 4.验证了胶原蛋白修饰的<ol><li>制备1 ml的样品从每个本地，甲基化，以及琥珀酰化的胶原蛋白溶液稀释到浓entration 0.1毫克/毫升，用无菌纯水的氢离子滴定。进一步稀释缓冲至少1000倍通过用水透析通过10kDa的截止membraneusing 3溶液的变化对于每个至少4小时。 </li><li>调节溶液的pH至7.3与少量的NaOH和HCl的。用pH 7.3作为任意的基准，由16 Tanford如所述创建滴定曲线用于母体，甲基化，以及琥珀酰化胶原溶液在每个样品进行氢离子滴定。 </li><li>绘制在pH变化每酸的体积加入与结合的H +的每个分子的离子数。高pH值"氨基"的范围应显示在琥珀酰化的胶原蛋白的移位朝中性点（胺基团的损失），并且低pH值"羧基"的范围应显示在甲基化胶原左移（羧基基团的损失），并用琥珀酰化的胶原右移（在羧基的增益多个），相比于天然胶原。 </li><li>通过测定氨基的％在天然胶原使用2,4,6-三硝基苯磺酸（TNBA）比色法由琥珀酰化取代，下列标准协议17,18评估琥珀酰化反应的功效。 </li></ol> 5.制造微流体装置和细胞接种<ol><li>编造使用标准方法9的微流体装置。使用PDMS从SU-8大师副本成型硅使用光刻制造微流体细胞培养室，100微米高，0.4-1.5毫米宽，1〜10毫米长的通道，细胞生长定义。 </li><li>使用等离子体清洁器以氧化装置和载玻片的表面上，然后按共同键。通过暴露杀菌设备在UV光下至少30分钟后，填充腔室50微克/毫升纤连蛋白在无菌PBS孵育在37℃下45分钟。 </li&gt; <li>种子设备用的细胞，如原代大鼠或人类肝细胞，这需要对表型或分化状态的稳定胶原凝胶。我们使用20微升新鲜分离原代大鼠肝7,19或市售冷冻保存的原代人肝细胞以14×10 6个细胞/每毫升设备。 </li><li>允许细胞附着4-6小时，然后洗出独立的细胞和生长介质取代电镀媒体。孵育O / N，以确保充分细胞扩散，创造细胞的融合单层。 </li></ol> 6.层 - 层胶原沉积<ol><li>在一个层流组织培养罩，准备足够的卷对每个器件的各溶液10应用甲基化和琥珀酰化的胶原蛋白溶液，以及媒体几毫升。置于冰上的解决方案。我们用20微升胶原蛋白（10〜15倍，总装置容量）每一层的每个设备。 </li><li>是轧花与甲基化（聚阳离子）溶液，交替与20μl的甲基化，然后冲洗设备琥珀酰化的胶原蛋白溶液，等待每个应用之间1分钟。冲洗设备总共每溶液10次，这大约需要20分钟。加速工作，以最大限度地减少时间的细胞是没有介质的数量。 </li><li>观察胶原慢慢聚集在入口/出口取决于其大小。如果流体流的阻力增加，冲洗装置一次或两次的介质，然后继续分层。 </li><li>施加所有的层之后，冲洗装置两次用新鲜培养基并返回到培养箱中。取决于细胞类型，所述细胞外基质诱导形态学变化，如在肝细胞中增强的偏振，应是在几个小时内可见。 </li><li>使用标准方法9来验证胶原基质的存在下制备用于透射电子显微镜代表性装置装配上培养的细胞的顶端。 </li></ol> 7.稳定细胞表型和功能的<ol><li>图像的细胞形态，活力，和极性使用标准方法的细胞类型。对于肝细胞，采集图像上使用相差显微镜，LIVE / DEAD染色的可行性，并CMFDA染料心尖极化以证明胶原沉积的影响14天。 </li><li>对于细胞形态，用移液管通过入口冲洗装置与20μl的PBS漂洗，注入20微升PBS，并使用图像相差显微镜设备。 </li><li>对活/死染色，通过入口用移液管与20μl的PBS漂洗，注入20微升用DAPI LIVE / DEAD染色溶液制备下列制造商的说明冲洗装置中，温育30分钟，在37℃，冲洗设备再次用20微升PBS，和图像用荧光显微镜的装置。 </li><li>对于胆汁CANALIculi染色，用移液管通过入口冲洗装置与20μl的PBS漂洗，注入20微升2用DAPIμMCMFDA染色溶液制备按照生产商的说明，孵育30分钟，在37℃，再次冲洗装置20微升PBS，和图像用荧光显微镜的装置。 </li><li>收集用过的培养基和测量细胞代谢产物在适当的时间点在培养持续时间，以确定细胞的功能。对于肝细胞，测量在用过的培养基白蛋白和尿素的量。 </li><li>上的细胞本身，例如评估酶活性的水平，抗体染色，或RNA分析执行对端点功能分析。对于肝细胞，诱导和测量细胞色素P450酶的活性或II期缀合的酶谷胱甘肽S-转移酶。 </li></ol>

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The authors have nothing to disclose.

超薄纯胶原组件可以沉积在使用改性胶原的层 - 层淀积带电的细胞或材料的表面。这项研究的结果表明，甲基化和天然胶原的琥珀酰化创建聚阳离子和聚阴离子胶原溶液（图1）可与层-层技术用于沉积超薄胶原基质组件上的细胞（ 图2）或其它带电材料表面。这种超薄基质层可以稳定形态，存活率，和偏振细胞如肝细胞（图3），播种在微流体装置，以及它们的功能之后（图4）。 对于超薄胶原层的成功沉积，改性反应创建甲基和琥珀酰化胶原是至关重要的。因为羧基甲基化反应是不利的，则胶原甲基化反应必须在酸化的甲醇中进行，以驱动反应向前，根据勒夏特列原理。因此，从胶原pH值沉淀后，为了提高反应效率溶解沉淀的胶原蛋白在酸化甲醇中之前除去所有过量的水是很重要的。为琥珀酰化反应，保持溶液的pH值高于9同时加入琥珀酸酐的丙酮是至关重要的。连续监测pH并缓慢加入琥珀酐丙酮溶液的是确保进行有效的反应有帮助的。在层 - 层沉积，关键是要尽量减少时间使细胞去无介质。 10双层的过程需要大约20分钟，大约是只要大多数细胞应该没有媒体的保育箱的外侧。如果超过10双层必须存入，休息中的细胞培养基在培养箱中科拉之间3-4小时根沉积，以确保细胞的健康。在另一个极端，太少层不会有效地维持细胞形态。在我们的测试中，3层并没有维持细胞的形态，而10层做到了。 若干修改可以作出的程序来解决问题。时间和甲基化反应的温度可以在出现问题时进行改变。例如，降低反应温度至4℃，并增加时间7天可能提高效率。另外，根据聚合的pH提取期间的范围内，胶原有时会完全甲基化反应过程中溶解在酸化的甲醇中。如果发生这种情况，加10 M氢氧化钠以使pH达9-10后的反应，以便沉淀出胶原蛋白，以便它可以在下一步骤中被沉淀已完成。同时施加所述层 - 层沉积在微流体装置，小通道或端口可以成为厘ogged与胶原蛋白，通过增加该装置的流体阻力来表示。冲洗设备用新鲜媒体应消除任何障碍，使沉积的继续。 如这里所描述的层 - 层的胶原的手册沉积是在微流体装置的缩放和自动化方面的限制。然而，这种技术与标准的微流体的硬件，包括阀和泵可用于自动化的技术中，并在一次准备设备的阵列兼容。这种技术用于平板培养使用的另一个限制是，它需要相对大量天然胶原的。微流体应用，甲基化和琥珀酰化胶原的体积可以用于创建在很多设备超薄胶原层。用标准的组织培养板，另一方面，需要涂覆的孔的容积变得更加令人望而却步随阱大小。 这层 -由层技术的超薄胶原基质层的沉积可以通过使用替代的或功能上的修改，以及通过创建细胞由薄矩阵组件分离的多个层得到进一步的发展。虽然甲基化和琥珀酰化的相对简单的氨基酸侧链改性反应均用在这里，可替代的或额外的反应可以被用来代替，以创建官能胶原。官能化侧链应该仍然产生的蛋白质与整体正负电荷净，但是这样的官能化可以是用于各种应用是有用的。

Although microfluidics allows for the exquisite control of the cellular microenvironment, culturing cells, especially primary cells, within microfluidic devices can be challenging. Many traditional cell culture techniques have been developed to sustain and stabilize cell function when cultured in tissue culture plates, but translating those techniques to microfluidic devices is often difficult. 
One such technique is the culture of cells on or sandwiched between collagen gels as a model of the physiological 3D cell environment.1 Type I collagen is one of the most frequently used proteins for biomaterials applications because of its ubiquity in extracellular matrix, natural abundance, robust cell attachment sites, and biocompatibility.2 Many cells benefit from 3D culture with collagen, including cancer cells3,45, microvascular endothelial cells6, and hepatocytes7, among others. While the use of collagen gels is easy in open formats, such as tissue culture plates, the limited channel dimensions and enclosed nature of microfluidic devices makes the use of liquids that gel impractical without blocking the entire channel.
To overcome this problem, we combined the layer-by-layer deposition technique8 with chemical modifications of native collagen solutions to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These layers can stabilize cell morphology and function similar to collagen gels and can be deposited on cells in microfluidic devices without blocking the channels with polymerized matrix. The goal of this method is to modify native collagen to create polycationic and polyanionic collagen solutions and to stabilize cells in microfluidic culture by depositing thin collagen matrix assemblies onto the cells. This technique has been used to stabilize the morphology and function of primary hepatocytes in microfluidic devices.9
Although layer-by-layer deposition has previously been reported with natural and synthetic polyelectrolytes10 to cover hepatocytes in plate culture11,12 and as a seeding layer for hepatocytes in microfluidic devices13,14, this method describes the deposition of a pure collagen layer on top of hepatocytes, mimicking the 3D collagen culture techniques. In this protocol, we use hepatocytes as example cells that can be maintained using 3D collagen layers. The many other types of cells that benefit from 3D culture in collagen may similarly benefit from culture after layer-by-layer deposition of an ultrathin collagen matrix assembly.

<table><tbody><tr><td>collagen type I, rat tail</td><td>Life Technologies</td><td>A1048301</td><td>option for concentrated rat tail collagen</td></tr><tr><td>collagen type I, rat tail</td><td>Sigma-Aldrich</td><td>C3867-1VL</td><td>option for concentrated rat tail collagen</td></tr><tr><td>collagen type I, rat tail</td><td>EMD Millipore</td><td>08-115</td><td>option for concentrated rat tail collagen</td></tr><tr><td>collagen type I, rat tail</td><td>R%D Systems</td><td>3440-100-01</td><td>option for concentrated rat tail collagen</td></tr><tr><td>succinic anhydride</td><td>Sigma-Aldrich</td><td>239690-50G</td><td>succinylation reagent</td></tr><tr><td>anhydrous methanol</td><td>Sigma-Aldrich</td><td>322415-100ML</td><td>methylation reagent</td></tr><tr><td>sodium hydroxide</td><td>Sigma-Aldrich</td><td>S5881-500G</td><td>pH precipitation reagent</td></tr><tr><td>hydrochloric acid</td><td>Sigma-Aldrich</td><td>320331-500ML</td><td>pH precipitation reagent</td></tr><tr><td>rat collagen type I ELISA</td><td>Chondrex</td><td>6013</td><td>option for detecting collagen content</td></tr><tr><td>hydroxyproline assay kit</td><td>Sigma-Aldrich</td><td>MAK008-1KT</td><td>option for detecting collagen content</td></tr><tr><td>hydroxyproline assay kit</td><td>Quickzyme Biosciences</td><td>QZBtotcol1</td><td>option for detecting collagen content</td></tr></tbody></table>

layer-by-layer collagen deposition in microfluidic devices for microtissue stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

天然胶原可以使用甲基化和琥珀酰化创建用于在层 - 层沉积用聚阳离子和聚阴离子胶原溶液进行修改。琥珀酰化修饰天然胶原的ε-氨基与琥珀基团，和甲基化修饰天然胶原的羧基与甲基（图1A）。这些修改到胶原蛋白的氨基酸侧链改变pH滴定曲线的解决方案。琥珀酰化降低氨基团的数目，增加羧基基团的数目，而甲基化对氨基没有影响，并减少羧基基团的数目，相比于天然胶原（图1B）。这些修饰改变胶原分子的电荷特征。琥珀酰化除去带正电荷的基团，并将它们替换负电荷的基团，和甲基化去除带负电荷的基团（图1C &lt;/ STRONG&gt;）。 甲基化和琥珀酰化的胶原蛋白溶液（COL LBL）的层-层沉积上创建电荷的表面，如细 ​​胞 （图2A）的顶部的超薄胶原基质组件。一个微流体装置（图2B）内接种于纤维连接蛋白包被的玻璃原代肝细胞可涂覆有这样的纯粹的胶原基质组件（图2C）。 10双层对细胞沉积产生的大约140纳米（ 图2D）的胶原层的厚度。 使用COL LBL技术沉积的超薄胶原蛋白聚集层稳定原代肝细胞超过14天，微流体装置。肝细胞没有顶部矩阵罩失去其分化表型，合同和抬离的微流体装置的表面上随时间（图3A-C）。与此相反，覆盖着超薄的胶原蛋白组装maintai肝细胞ñ其分化的形态和偏振超过14 天 （图3D-F）。细胞的生存力保持在大于90％（图3G-1）。此外，与COL LBL处理的肝细胞的偏振随时间增加，表示心尖胆汁小管网络 （图3J-L）的发展。 除了细胞活力，形态和偏振，所述COL LBL技术恢复并稳定原代肝细胞中的类似的细胞板的标准技术水平的功能。白蛋白由肝细胞分泌低下电镀后，并保持低电平，除非细胞被诱导极化通过矩阵覆盖。后COL LBL治疗剂，稳定剂（图4A）前白蛋白分泌由肝细胞的增加超过8-10天。尿素生产细胞接种后立即高，随时间下降的电池NOT覆盖有基质，后在肝细胞中稳定化COL LBL（图4B）8-10天尿素生产稳定。细胞色素P450（CYP）响应于刺激酶表达是肝细胞中的一个专门的功能。 CYP1A1在肝细胞的诱导响应于3-甲基胆回收并稳定长达14天由COL LBL治疗（图4C）。 <img alt="图1" src="/files/ftp_upload/53078/53078fig1.jpg" /> 图1.鼠尾胶原溶液化学修饰，以积极创造净利润或净负电荷的高分子溶液。 （A）中的琥珀酰化和甲基化反应，修改羧基和ε氨基组分别的示意图。（B）中示出的H +离子以改变关口后溶液的pH值所需要的相对数量移位氢滴定曲线衍生物拉根修饰。（C）的净电荷（平均值±SD）从H +轮换着算出的改性胶原溶液（B）的标准化为天然胶原的。 MC:甲基化的胶原蛋白。 SC:琥珀胶原蛋白。 AA:氨基酸残基。重新打印许可的9。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53078/53078fig1large.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/53078/53078fig2.jpg" /> 图2.层-层改性的胶原溶液沉积在肝细胞的顶端产生的超薄胶原基质组件。 （A）中的肝细胞接种于纤维连接蛋白上的微型装置和温育具有交替的甲基化和琥珀酰化的胶原蛋白溶液的示意图。 TEM横截面代表（20个细胞每组检查）CON控制（B）和 COL LBL处理后第2天的培养（三）肝细胞。箭头:LBL胶原层。比例尺:2微米（D）COL LBL层每从TEM照片（N = 20个细胞的4个设备）来测量电池厚度（平均值±SD）。从9修改。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53078/53078fig2large.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/53078/53078fig3.jpg" /> 图3. COL LBL技术，文化保持肝细胞形态，活力，并在微器件极化超过14天，肝细胞，没有顶层的微型器件（阴性对照）合同，并抬离地面随着时间的推移（A-C）。改性胶原的LBL沉积维持肝细胞形态（D-F）和VIABility（G-I）的微型器件超过14天。 CMFDA是一个通用的细胞标记保留在非极化肝细胞（J）的细胞质中。随着时间的推移，胆小管的发展可以通过荧光团进入泪小管空间（K）的周围的细胞随着时间的发展（L）的排泄可以看出。图片比例尺:100微米。插图比例尺:50微米。从9修改。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53078/53078fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="Figure4" src="/files/ftp_upload/53078/53078fig4.jpg" /> 图4. COL LBL稳定肝白蛋白和尿素的部分和CYP活性超过在微流体装置14天。（A）的白蛋白分泌由COL LBL肝细胞开始低和随时间增加ùNTIL在第10天（B）的尿素分泌COL LBL肝细胞达到平台随时间降低直至达到平台在第10天（C）的 CYP1A活性的诱导低，为2天，但在第7天显著增加，一这是经过14天平均值±SEM保持水平; COL LBL:肝细胞LBL胶原沉积后培养的; NoTop:肝细胞无顶基质层培养; N = 6-8的设备。从9修改。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53078/53078fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization at JoVE.com

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

层与层胶原沉积在微流体装置的Microtissue稳定

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

layer-layer-collagen-deposition-microfluidic-devices-for-microtissue

Nanyang Technological University

Research

JoVE Journal

Bioengineering

9.3K 次观看.  Massachusetts General Hospital, Harvard Medical School. The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

视频: Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized tissues.  The modules can be made of collagen as well as other gelable or crosslinkable materials.  They are approximately 2 mm in length and 0.7 mm in diameter upon fabrication but shrink in size with embedded cells or when the modules are coated with endothelial cells.  The modules individually are small enough that the embedded cells are within the diffusion limit of oxygen and other nutrients but modules can be packed together to form larger tissues that are perfusable.  These tissues are modular in construction because different cell types can be embedded in or coated on the modules before they are packed together to form complex tissues.  There are three main steps to making the modules: (1) neutralizing the collagen and embedding cells in it, (2) gelling the collagen in the tube and cutting the modules and (3) coating the modules with endothelial cells.

Creation of micro-tissues using cylindrical collagen gels, called modules, that contain embedded cells and which surface is coated with endothelial cells.

微组织使用胶原蛋白的模块的制备凝胶含有细胞

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized ...

科研

生物工程

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

微机械动态细胞培养平台

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

器官Ⅰ型胶原的含量可延展的平台，以评估细胞行为的一个3维的情况下

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

工程三维细胞化胶原凝胶的血管组织再生

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular behaviors on disease-relevant time scales. Previously, microcontact printing has been used to fabricate two-dimensional functional arterial mimics through patterning of extracellular matrix protein as guidance cues for tissue organization. Vascular muscular thin films utilized these mimics to assess functional contractility. However, the microcontact printing fabrication technique used typically incorporates hydrophobic PDMS substrates. As the tissue turns over the underlying extracellular matrix, new proteins must undergo a conformational change or denaturing in order to expose hydrophobic amino acid residues to the hydrophobic PDMS surfaces for attachment, resulting in altered matrix protein bioactivity, delamination, and death of the tissues.
Here, we present a microfluidic deposition technique for patterning of the crosslinker compound genipin. Genipin serves as an intermediary between patterned tissues and PDMS substrates, allowing cells to deposit newly-synthesized extracellular matrix protein onto a more hydrophilic surface and remain attached to the PDMS substrates. We also show that extracellular matrix proteins can be patterned directly onto deposited genipin, allowing dictation of engineered tissue structure. Tissues fabricated with this technique show high fidelity in both structural alignment and contractile function of vascular smooth muscle tissue in a vascular muscular thin film model. This technique can be extended using other cell types and provides the framework for future study of chronic tissue- and organ-level functionality.

We present a method for microfluidic deposition of patterned genipin and fibronectin on PDMS substrates, allowing extended viability of vascular smooth muscle cell-dense tissues. This tissue fabrication method is combined with previous vascular muscular thin film technology to measure vascular contractility over disease-relevant time courses.

微京尼平沉积技术的微模式化血管肌肉薄膜的扩展文化

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular ...

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 ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

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 ...

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.

微流控生物工程血管化组织和细胞器解体

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth plate cartilage architecture and results in growth modulation of long bones of children. To determine the role of compressive stress in bone growth, we created a microfluidic device actuated by pneumatic pressure, to dynamically (or statically) compress growth plate chondrocytes embedded in alginate hydrogel cylinders. In this article, we describe detailed methods for fabricating and characterizing this device. The advantages of our protocol are: 1) Five different magnitudes of compressive stress can be generated on five technical replicates in a single platform, 2) It is easy to visualize cell morphology via a conventional light microscope, 3) Cells can be rapidly isolated from the device after compression to facilitate downstream assays, and 4) The platform can be applied to study mechanobiology of any cell type that can grow in hydrogels.

This article provides detailed methods for fabricating and characterizing a pneumatically actuating microfluidic device for chondrocyte compression.

微流体平台，用于用动态压缩刺激软骨细胞

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth ...

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart muscle, the myocardium, is notoriously difficult to accomplish in vitro. Mainly, obstacles lie in the need for human cardiomyocytes (CMs) that are either adult or exhibit adult-like phenotypes and can successfully replicate the myocardium&#39;s cellular complexity and intricate 3D architecture. Unfortunately, due to ethical concerns and lack of available primary patient-derived human cardiac tissue, combined with the minimal proliferation of CMs, the sourcing of viable human CMs has been a limiting step for cardiac tissue engineering. To this end, most research has transitioned toward cardiac differentiation of human induced pluripotent stem cells (hiPSCs) as the primary source of human CMs, resulting in the wide incorporation of hiPSC-CMs within in vitro assays for cardiac tissue modeling.
Here in this work, we demonstrate a protocol for developing a 3D mature stem cell-derived human cardiac tissue within a microfluidic device. We specifically explain and visually demonstrate the production of a 3D in vitro anisotropic cardiac tissue-on-a-chip model from hiPSC-derived CMs. We primarily describe a purification protocol to select for CMs, the co-culture of cells with a defined ratio via mixing CMs with human CFs (hCFs), and suspension of this co-culture within the collagen-based hydrogel. We further demonstrate the injection of the cell-laden hydrogel within our well-defined microfluidic device, embedded with staggered elliptical microposts that serve as surface topography to induce a high degree of alignment of the surrounding cells and the hydrogel matrix, mimicking the architecture of the native myocardium. We envision that the proposed 3D anisotropic cardiac tissue-on-chip model is suitable for fundamental biology studies, disease modeling, and, through its use as a screening tool, pharmaceutical testing.

The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.

在微流体平台内开发 3D 组织人类心脏组织

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart ...