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?...

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 border="0" cellpadding="0" cellspacing="0">
	<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?...

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

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.