这项工作得到了 JST (JPMJCR14W4)、促进科学协会 (jsp) KAKENHI (赠款编号25600060、16K16386)、下个和 JST 创新项目中心的支持, 项目重点是开发关键的评估技术, 从日本医药研发机构, 阿曼德, 瑞穗科技促进会。《京都大学纳米技术中心》支持微细加工。

1. 微流控装置模具的制作
<ol><li>使用商用软件 (Clewin5 或 AutoCAD 2016,等) 设计微流控设备的模式。有关 Clewin5 的功能, 请参阅用户手册 (http://manualzz.com/doc/7159150/clewin-user-s-manual)。 注意: 设计文件在辅助文件 1中可用。</li>
	<li>将设计文件传送到微阵列生成器, 并将该工具加载到涂有正胶的铬罩上。</li>
	<li>使用微阵列发生器在图案区域中暴露正光刻胶。</li>
	<li>使用开发人员 (材料表) 开发正光刻胶, 并用去离子 (DI) 水冲洗面罩。</li>
	<li>使用铬蚀刻 (材料表), 蚀刻去除正胶的暴露区域中的铬。用底水冲洗面罩。</li>
	<li>使用丙酮去除掩膜上剩余的光刻胶。 注意: 透明掩码可以替代 Cr 掩码。</li>
	<li>准备一个清洁的硅片 (4 英寸, P (100)) 和自旋涂层 hexamethyldisilazane (HMDS) 在3000转每分钟三十年代。</li>
	<li>Softbake HMDS 5 分钟, 120 摄氏度, 室温下将晶片冷却5分钟。</li>
	<li>Spincoat 负光刻胶 (材料表) 在十年代和 1200 rpm 为三十年代的 500 rpm。 注: 自旋涂层条件应产生的光刻胶层厚度约100µm 在晶片上光刻后。</li>
	<li>在室温下, 焙45分钟的负光刻胶, 在95摄氏度时冷却晶片60分钟。</li>
	<li>检查每个实验中的 UV 光强度, 并计算总曝光能量剂量在250兆瓦/厘米2上的曝光时间。将光掩模 (步骤 1.1-1.6) 放在晶片上, 并将其暴露在 UV 光上。</li>
	<li>Postbake 负光刻胶为1分钟, 在65摄氏度和5分钟95摄氏度。允许晶片在室温下冷却1分钟。</li>
	<li>在第一个开发者 (材料表) 浴缸和第二个浴缸中的2分钟内, 开发15分钟的负光刻胶层。在十年代的第一个异丙醇 (ipa) 浴中漂洗硅片, 在十年代的第二个 ipa 浴中冲洗。</li>
	<li>在室温下, Hardbake 30 分钟的负光刻胶, 在200摄氏度时冷却晶片5分钟。</li>
	<li>使用曲面探查器测量负光刻胶层的厚度。</li>
	<li>将晶片放在连接到真空泵的干燥中, 加入200µL 硅烷 (氯 (1 h、1 h、2 h、2 h perfluorooctyl) 硅烷)。打开泵10分钟, 然后关闭, 并保持晶片在干燥4小时。</li>
</ol>2. 制造步骤和组装
<ol><li>铸造烷 (聚硅烷) 预聚合物 (基部: 固化剂 = 10:1 (w/瓦)) 和德加在真空室为 2 h。</li>
	<li>一夜之间在一个空气排出的烤箱里治疗80°c。</li>
	<li>从硅片上剥离出硅烷。</li>
	<li>打孔 1A-3B (图 1) 和球体文化井。使用2毫米直径冲孔 1A-3B, 和一个1毫米直径打孔的球体井。</li>
	<li>通过反复粘接和剥离胶带, 用胶带清洁该板和玻璃罩滑动 (24 毫米 x 24 毫米)。然后在四十年代 (40 兆瓦, 50 sccm) 上用空气等离子体处理该板。</li>
	<li>将该板粘接在玻璃盖上, 以从表面上排出的任何气泡, 并在80摄氏度至少12小时内消除。</li>
</ol>3. 球体准备
注: 在本研究中, 用红荧光蛋白表达人脐静脉内皮细胞 (RFP-血管内皮细胞) 和绿色荧光蛋白表达血管内皮细胞 (GFP-血管内皮细胞) 分别用于球体和微, 以区分起源的血管内皮细胞后建立了 perfusable 血管网。如果不需要血管内皮细胞的起源, 未标记的血管内皮细胞就足以进行实验。
<ol><li>解冻的 RFP-血管内皮细胞 (3.0 x10 5 细胞/瓶) 和人肺成纤维细胞 (hLF) (1.0 x10 6 细胞/小瓶), 并添加到10毫升的培养基为内皮细胞和成纤维细胞, 分别(表的材料)。在37摄氏度和 5% CO2的100毫米菜肴中培养它们, 用于分汇合 RFP-血管内皮细胞和 hLFs 2-3 天。这项准备将产生200球体。</li>
	<li>用2毫升0.05% 胰蛋白酶-EDTA 分离血管内皮细胞和 hLFs, 停止胰蛋白酶反应, 4 毫升的 DMEM 含有 10% (v/v) 胎牛血清 (血清) 和 1% (v/v) 青霉素/链霉素。</li>
	<li>离心后在 220 x g 3 分钟, 移除上清, 并并用重悬在内皮介质中的 RFP 血管内皮细胞和 hLFs 到最终细胞浓度分别为2.5 x 10 4 和1.0 x 10 5 细胞/毫升。</li>
	<li>在具有超低结合面的96井板上, 轻轻地添加200µL 的细胞悬浮液 (5000 细胞用于 RFP-血管内皮细胞和2万细胞 hLFs)。</li>
	<li>在37°c 和 5% CO2上孵化96井板, 为期2-4 天。 注: 由于球体直径取决于96井板中的培养周期和初始细胞数, 因此可以由这两个参数控制。</li>
</ol>4. 微流控装置中的细胞播种
注意: 孔、通道和球体井的命名约定显示在图 1中。我们将0天定义为在微流控装置中完成细胞采集的那一天。实验时间线的示意图显示在图 2中。
<ol><li>天−1) 球体装载 注: 应在冰上执行以下步骤, 以防止胶原蛋白和纤维蛋白的凝胶化。<ol>
<li>在磷酸盐缓冲生理盐水 (PBS) 中溶解纤维蛋白原, 最终浓度为2.80 毫克/毫升。</li>
		<li>根据制造商的协议, 在 PBS 中制备中性胶原蛋白 (3.0 毫克/毫升)。</li>
		<li>结合107.2 µL 的纤维蛋白原溶液 (2.80 毫克/毫升), 8.0 µL 的中和胶原蛋白 (3.0 毫克/毫升) 和3.6 µL 的抑肽酶 (5 U/毫升) 每反应在一管。此解决方案被标记为 "主混合解决方案" (MS)。 注: 体积足够装载一个球体。如果有多个球体, 则将每个卷乘以球体的数目。</li>
		<li>在 PBS 中溶解凝血酶, 最终浓度为50毫升。 注:50 毫升凝血酶 (20 µL/管) 的整除数储存在−30°c, 在每次试验前解冻。</li>
		<li>将含有球体的96井板放在生物安全柜内。</li>
		<li>在生物安全柜内准备两个35毫米的培养皿, 一盘放在冰上 (碟 1), 另一盘放在台式 (盘子 2) 上。吸管99µL 的 MS 在盘 1 (图 3a) 的中心, 形成一个水滴。</li>
		<li>修剪2黄色 (10-100 µL) 和3个明确的吸管提示 (1-100 µL) 的提示, 收集球体从96井板, 混合凝血酶与 MS, 精确收集球体, 向设备注入球体, 并向设备注入介质分别.尖端的孔径应该比直径 1A-3B 通道1和3或球体井在2通道的舒适适合。 注: 此后, 除非另行注明, 使用此步骤中切割的吸管提示。剪切提示的照片在图 4中可用。</li>
		<li>从96井板中收集100µL 的球体, 并将其添加到盘 2 (图 3a)。</li>
		<li>从碟2中提取出最小的介质数量的球体。当 pipettor 直立时, 球体应该通过重力向吸管尖端的底部移动。通过触摸吸管尖到碟 1 (图 3a) 的 MS 雾滴的半月板上, 弹出球体。 注意: 应快速执行以下步骤 4.1.10 & 4.1.11。</li>
		<li>添加1µL 凝血酶 (50 U/毫升), 并轻轻地与黄色吸管提示混合。</li>
		<li>随着吸管设置为7µL, 捡起球体, 慢慢地把它放进球体井 (图 3b)。</li>
		<li>在37摄氏度孵育15分钟, 用于纤维蛋白的凝胶化。</li>
		<li>慢慢地从1A 和3A 孔中注入内皮介质, 用介质 (20 ~ 30 µL/通道) 填充通道1和3。</li>
		<li>将设备放在带有湿 Kimwipe 的100毫米盘中, 以防止介质从设备中蒸发 (图 3c)。</li>
		<li>将设备放置在37摄氏度和 5% CO2上, 24 h 以去除介质和纤维蛋白之间界面上的气泡。</li>
	</ol>
</li>
	<li>0天) 血管内皮细胞装载<ol>
<li>解冻 GFP-血管内皮细胞 (3.0 x10 5 细胞/小瓶), 并添加到10毫升的内皮介质。在37摄氏度和 5% CO2的100毫米菜肴中培养它们, 2-3 天到达亚融合。一盘亚汇合的 GFP-血管内皮细胞是足够的准备8-10 设备。</li>
		<li>分离 GFP-血管内皮细胞从菜肴与2毫升0.05% 胰蛋白酶-EDTA 和停止胰蛋白酶反应与4毫升的 DMEM 含有 10% (v/v) 胎牛血清 (血清) 和 1% (v/v) 青霉素/链霉素。</li>
		<li>离心后在 220 x g 3 分钟, 并用重悬在内皮介质中的 GFP-血管内皮细胞在5.0 x 10 6 细胞/毫升。</li>
		<li>将血管内皮细胞细胞悬浮液注入通道1通孔 1B (20 µL/通道)。</li>
		<li>倾斜的微流控装置 90°, 放置在侧面和孵化37°c 30 分钟, 以确保血管内皮细胞坚持在2通道的纤维蛋白。<ol>
<li>确认血管内皮细胞对纤维蛋白的附着。当在凝胶和培养基之间的接口上连接的血管内皮细胞数不够时, 在设备培养几天后, 血管根部可能会断开连接(图 5)。在该协议中, 灌注的成功率为 > 50%。</li>
		</ol>
</li>
		<li>对通道3重复步骤4.2.4 和4.2.5。</li>
		<li>100毫米菜肴中的培养细胞, 湿度 Kimwipe 在37摄氏度和 5% CO2 7-14 天。</li>
	</ol>
</li>
	<li>1天 ~) 媒体交流<ol>
<li>每天在频道1和3中交换一半的媒体。</li>
	</ol>
</li>
</ol>5. 细胞核染色
<ol><li>在 PBS 准备4% 多聚甲醛 (粉煤灰)。</li>
	<li>从通道1和3中移除介质, 并在每个通道中添加20µL 4% 粉煤灰。要更换4% 粉煤灰中的溶液, 请重复从设备中移除该溶液, 并增加4% 粉煤灰三倍。</li>
	<li>在一夜之间孵化4摄氏度的装置。</li>
	<li>从设备中取出4% 个粉煤灰, 用 PBS 清洗通道三次。</li>
	<li>添加20µL 10 µg/毫升的荧光染料 (材料表) 每通道染色细胞细胞核。要在设备中交换解决方案, 请重复从设备中删除该解决方案, 并添加10µg/毫升荧光染料三次。</li>
	<li>将设备存储在4摄氏度, 24 小时。</li>
</ol>6. 球体的流体灌注
<ol><li>在37摄氏度和 5% CO2的设备中培养超过7天的球体, 以完成连续的血管通路。</li>
	<li>从1A、1B、3A 和3B 孔中取出介质。</li>
	<li>介绍10µL 10 µM 溶液的荧光素异硫氰酸酯 (FITC)-葡聚糖在 PBS 入孔1A 和1B。</li>
	<li>在倒置显微镜下监测微珠或 FITC 染料的流动情况。</li>
</ol>7. 芽长的量化
注: 本研究 ImageJ 1.49 软件用于图像分析。
<ol><li>重叠的图像 GFP-血管内皮细胞天 0, 1, 3, 5 和7。</li>
	<li>在1、3、5和7天拍摄的图像中, 从同一位置减去0天的血管尖端的位置。</li>
	<li>从根位置确定血管尖端的生长 (图 6)。血管尖端和根之间的距离被定义为这项研究的萌芽长度 (图 7b)。</li>
</ol>8. 血管角度的量化
注意: 血管角度被定义为由血管角度、根和球体中心组成的角度 (图 7c)。
<ol><li>Binarize 共培养球体中含有 RFP 血管内皮细胞的荧光图像, 通过分析函数在 ImageJ 中测量 "质心" 位置。在本研究中, 测量的 "质心" 位置被定义为球体的中心 (图 6)。</li>
	<li>在步骤7中用同样的方法确定血管尖端和根部的位置。</li>
	<li>在 ImageJ 软件中使用分析函数测量血管角度。</li>
</ol>

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提交人宣布, 他们没有竞争的金融利益。

以前的报告表明, hLFs 分泌多种血管生成因子的鸡尾酒, 如 angiopoietin-1, 染, 肝细胞生长因子, 转化生长因子α, 肿瘤坏死因子和一些细胞外基质蛋白29, 30。这种检测依赖于共培养球体中 hLFs 的血管生成物分泌, 这是该技术的局限性。因此, 在井底设置一个共培养球体是稳定的血管形成的关键, 因此共培养球体与血管内皮细胞通道1和3之间的距离可以缩短。成?...

从单层 (二维) 文化转移到三维文化, 是因为需要使用模仿活体组织的细胞功能的文化模型1,2,3。细胞培养中常用的扁平和硬质塑料基底不像人体内大部分的胞外环境。事实上, 许多研究表明, 三维的文化重现了组织特异性的结构, 机械和生物化学的线索, 和细胞通讯, 没有在常规的二维文化中观察到4, 5,6,7,8。
多细胞聚合或球体, 是实现此三维区域性9、10的最有希望的技术之一。细胞分泌细胞外基质 (ECM), 可以与球体中的其他人相互作用。虽然一些其他生物工程方法11,12,13,14, 如单元格堆叠, 成功地复制人体的空间复杂性, 这些方法只有两个或三个种类的细胞, 便于分析, 只专注于一个功能的靶器官。相比之下, 球体中的细胞由于在球体中的养分、氧、分泌和分泌信号分子的异构供应而暴露在不同的文化环境中, 这取决于它们在球体中的位置。球体的此功能部分模仿体内区域性条件, 并使球体中的单元格在体外创建比堆叠组织9中培养的更复杂、组织化的组织结构.15,16. 请注意, 如果球体由单个细胞组成, 则球体中的细胞的功能由于球体中的异构环境而不均匀。在过去的几年中, 球体培养允许胚胎干细胞 (ESCs), 诱导多能干细胞 (iPSCs) 或组织驻留干细胞模仿体内发育序列, 并重新创建微型器官, 如大脑17,肝脏18和肾脏19,20。
尽管球形培养技术取得了重大进展, 但长期培养大型球体仍然存在问题。在一个三维的组织中, 细胞需要位于血管的150-200 µm 内, 因为氧气和营养素的供应有限21。在球体内的血管网络是必要的, 以重述血液和组织之间的交换物质在体内。为达到这一目的, 其他组与靶细胞共培养内皮细胞22,23,24或诱导多潜能细胞分化为 CD31-positive 细胞20。然而, 报告的船状结构没有腔的开放端向球体中心提供氧气和养分。为了模拟血管的作用, 滋养细胞在三维的文化, 开放和 perfusable 血管网络必须发展在球体。
在过去几年中, microengineering 领域的一些研究小组报告了通过利用共培养成纤维细胞的血管生成因子在微流控装置中自发形成 perfusable 血管网络的方法 25 ,26。这些血管网络与它们的体内对应物具有相似的形态学, 可以被环境因素重塑, 使它们适合于在球状培养中模仿血管功能。本协议的目的是使用微流控平台27在球体中构造一个 perfusable 的血管网络。微流控设备从先前报告的设备25中进行了修改, 以便可以合并球体。通过将球状成纤维细胞的血管生成分泌物定向到微的内皮细胞, 微与球体吻合, 形成 perfusable 血管网络。这种方法允许直接交付广泛的物质, 如荧光分子和微米级的珠子进入球体的内部, 这为长期组织培养与血管网络提供了框架。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AutoCAD 2017</td>
			<td>Autodesk</td>
			<td>AutoCAD 2017</td>
			<td></td>
		</tr>
		<tr>
			<td>A chromium mask coated with AZP 1350.</td>
			<td>CLEAN SURFACE TECHNOLOGY</td>
			<td>CBL2506Bu-AZP</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro pattern generator</td>
			<td>Heidelberg</td>
			<td>uPG101</td>
			<td></td>
		</tr>
		<tr>
			<td>MF CD-26 developer</td>
			<td>Rohm and haas electronic materials</td>
			<td>-</td>
			<td>Developer in protocol 1.4</td>
		</tr>
		<tr>
			<td>S-Clean</td>
			<td>Sasaki Chemical</td>
			<td>S-24</td>
			<td>Chromium etchant in protocol 1.5</td>
		</tr>
		<tr>
			<td>Aceton</td>
			<td>Wako</td>
			<td>012-00343</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon Wafer</td>
			<td>Canosis</td>
			<td>SiJ-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin Coater</td>
			<td>MIKASA</td>
			<td>1H-D7</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexamethyldisilazane (HMDS)</td>
			<td>Tokyo Ohka Kogyo</td>
			<td>H0089</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 3050</td>
			<td>MicroChem</td>
			<td>-</td>
			<td>Negative photoresist in protocol 1.9</td>
		</tr>
		<tr>
			<td>UV Exposure</td>
			<td>Nanometric Technology Inc</td>
			<td>LA310s</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 Developer</td>
			<td>MicroChem</td>
			<td>Y020100</td>
			<td>Developer for the negative photoresist in protocol 1.13</td>
		</tr>
		<tr>
			<td>2-propanol</td>
			<td>Wako</td>
			<td>163-04841</td>
			<td></td>
		</tr>
		<tr>
			<td>Surhace profiler</td>
			<td>Vecco</td>
			<td>Veeco Dektak XT-S</td>
			<td></td>
		</tr>
		<tr>
			<td>(Trichloro (1H, 1H, 2H, 2H-perfluorooctyl) silane</td>
			<td>Sigma</td>
			<td>448931</td>
			<td></td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS)</td>
			<td>Dow Corning Toray</td>
			<td>184W/C</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy Punch (1.0mm)&#160;</td>
			<td>Kai Industries</td>
			<td>BP-10F</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy Punch (2.0mm)&#160;</td>
			<td>Kai Industries</td>
			<td>BP-20F</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma System</td>
			<td>Femto Science</td>
			<td>COVANCE</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>MATSUNAMI GLASS</td>
			<td>C024241</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture Dishes</td>
			<td>Iwaki</td>
			<td>1000-035</td>
			<td></td>
		</tr>
		<tr>
			<td>RFP Expressing Human Umbilical Vein Endothelial Cell</td>
			<td>Angio Proteomie</td>
			<td>cAP-0001RFP</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Human Lung Fibroblasts</td>
			<td>Lonza</td>
			<td>CC-2512</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth Medium</td>
			<td>Lonza</td>
			<td>CC-3162</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibroblast Growth Media Kits</td>
			<td>Lonza</td>
			<td>CC-3132</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution</td>
			<td>Wako</td>
			<td>168-23191</td>
			<td></td>
		</tr>
		<tr>
			<td>0.05w/v% Trypsin-0.53mmol/l EDTA&#8226; 4Na Solution with Phenol Red</td>
			<td>Wako</td>
			<td>204-16935</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (Phosphate Buffered Salts)</td>
			<td>Takara bio</td>
			<td>T900</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Sumitomo bakelite</td>
			<td>631-21031</td>
			<td></td>
		</tr>
		<tr>
			<td>1000ul Chip</td>
			<td>NIPPON Genetics</td>
			<td>FG-402</td>
			<td></td>
		</tr>
		<tr>
			<td>200ul&#160; Chip</td>
			<td>NIPPON Genetics</td>
			<td>FG-301</td>
			<td></td>
		</tr>
		<tr>
			<td>10ul Chip</td>
			<td>NIPPON Genetics</td>
			<td>37650</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td>Model 370</td>
			<td></td>
		</tr>
		<tr>
			<td>GFP Expressing Human Umbilical Vein Endothelial Cell</td>
			<td>Angio Proteomie</td>
			<td>cAP-0001GFP</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibrinogen from bovine plasma</td>
			<td>Sigma</td>
			<td>F8630</td>
			<td></td>
		</tr>
		<tr>
			<td>Aprotinin from bovine lung</td>
			<td>Sigma</td>
			<td>A6279</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen I</td>
			<td>Corning</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin from bovine plasma</td>
			<td>Sigma</td>
			<td>T4648</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Invitrogen</td>
			<td>H21492</td>
			<td>Fluorescent dye to stain nuclei in protocol 5.5</td>
		</tr>
		<tr>
			<td>Paraformaldehyde Solution</td>
			<td>Wako</td>
			<td>163-25983</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Fluorescence Microscope</td>
			<td>OLYMPUS</td>
			<td>IX71</td>
			<td></td>
		</tr>
		<tr>
			<td>Degital CCD Camera</td>
			<td>OLYMPUS</td>
			<td>ORCA-R2</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Laser Scanning Biological Microscope</td>
			<td>OLYMPUS</td>
			<td>FV1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Fluorescence Microscope</td>
			<td>OLYMPUS</td>
			<td>IX-83</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate-dextran</td>
			<td>Sigma</td>
			<td>FD70S</td>
			<td></td>
		</tr>
	</tbody>
</table>

perfusable vascular network with a tissue model in a microfluidic device

球体 (多细胞聚合体) 被认为是人体内活体组织的良好模型。尽管在球体培养方面有了显著的进步, 球体的 perfusable 血管网仍然是维持和发展其功能所需的长期文化的关键挑战, 如蛋白质表达和形态发生。该协议提出了一种新的方法来集成 perfusable 血管网络在球体内的微流控装置。为了诱导球体中的 perfusable 血管网, 利用球形培养的人肺成纤维细胞的血管生成因子, 引导与微相连的血管生成苗。血管生成苗达到球形, 与球体中的内皮细胞合并, 形成连续的血管网络。血管网可以灌注球体内部, 而不会有任何渗漏。构建的血管网络可进一步用作营养物质的供应和废弃物的去除, 模拟血液循环在体内。该方法为球形培养提供了一个新的平台, 可以更好地对活体组织进行重述。

图 1显示了微流控设备的设计和照片。它有三个平行的渠道, 其中渠道2包含球形井。通道1和3用于 HUVEC 文化, 通道2为球体。每个通道由梯形 microposts 分离, 设计为模式。microposts 防止2通道中的水凝胶通过表面张力泄漏到通道1和 3, 允许在微28中的球体和血管内皮细胞之间交换物质。
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Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

微流控装置中组织模型的 Perfusable 血管网

该协议描述了如何在球体中设计一个 perfusable 的血管网。球体周围的微环境被设计来诱导血管生成, 并将球体连接到微流控装置中的微。该方法允许球状的灌注, 这是一个期待已久的技术在三维文化。

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

perfusable-vascular-network-with-tissue-model-microfluidic

Research

JoVE Journal

Bioengineering

13.8K 次观看.  Kyoto University. 该协议描述了如何在球体中设计一个 perfusable 的血管网。球体周围的微环境被设计来诱导血管生成, 并将球体连接到微流控装置中的微。该方法允许球状的灌注, 这是一个期待已久的技术在三维文化。

视频: Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

微流控装置重新创建一个肿瘤微环境<em在体外</em

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

科研

生物工程

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

一种微流体装置研究多个不同的菌株

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

预设计尺寸和形状的聚合物纤维和生物贿赂纤维的微流体制造

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

协议生物膜形成的流光在微流体器件与微柱

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren system is constructed from a Hoffman modulation contrast microscope, which provides easy access to the rear focal plane of the objective lens, by removing the slit plate and replacing the modulator with a knife-edge. The working principle of microscale schlieren technique relies on detecting light deflection caused by variation of refractive index1-3. The deflected light either escapes or is obstructed by the knife-edge to produce a bright or a dark band, respectively. If the refractive index of the mixture varies linearly with its composition, the local change in light intensity in the image plane is proportional to the concentration gradient normal to the optical axis. The micro-schlieren image gives a two-dimensional projection of the disturbed light produced by three-dimensional inhomogeneity.
To accomplish quantitative analysis, we describe a calibration procedure that mixes two fluids in a T-microchannel. We carry out a numerical simulation to obtain the concentration gradient in the T-microchannel that correlates closely with the corresponding micro-schlieren image. By comparison, a relationship between the grayscale readouts of the micro-schlieren image and the concentration gradients presented in a microfluidic device is established. Using this relationship, we are able to analyze the mixing inhomogeneity from associate micro-schlieren image and demonstrate the capability of microscale schlieren technique with measurements in a microfluidic oscillator4. For optically transparent fluids, microscale schlieren technique is an attractive diagnostic tool to provide instantaneous full-field information that retains the three-dimensional features of the mixing process.

Herein, we describe a procedure that employs microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. Through calibration, distribution of concentration gradient can be derived from the micro-schlieren image.

由微型条纹分析技术在微流控设备混合不均匀

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren ...

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 ...

Custom-designed Laser-based Heating Apparatus for Triggered Release of Cisplatin from Thermosensitive Liposomes with Magnetic Resonance Image Guidance

Liposomes have been employed as drug delivery systems to target solid tumors through exploitation of the enhanced permeability and retention (EPR) effect resulting in significant reductions in systemic toxicity. Nonetheless, insufficient release of encapsulated drug from liposomes has limited their clinical efficacy. Temperature-sensitive liposomes have been engineered to provide site-specific release of drug in order to overcome the problem of limited tumor drug bioavailability. Our lab has designed and developed a heat-activated thermosensitive liposome formulation of cisplatin (CDDP), known as HTLC, to provide triggered release of CDDP at solid tumors. Heat-activated delivery in vivo was achieved in murine models using a custom-built laser-based heating apparatus that provides a conformal heating pattern at the tumor site as confirmed by MR thermometry (MRT). A fiber optic temperature monitoring device was used to measure the temperature in real-time during the entire heating period with online adjustment of heat delivery by alternating the laser power. Drug delivery was optimized under magnetic resonance (MR) image guidance by co-encapsulation of an MR contrast agent (i.e.,&#160;gadoteridol) along with CDDP into the thermosensitive liposomes as a means to validate the heating protocol and to assess tumor accumulation. The heating protocol consisted of a preheating period of 5 min prior to administration of HTLC and 20 min heating post-injection. This heating protocol resulted in effective release of the encapsulated agents with the highest MR signal change observed in the heated tumor in comparison to the unheated tumor and muscle. This study demonstrated the successful application of the laser-based heating apparatus for preclinical thermosensitive liposome development and the importance of MR-guided validation of the heating protocol for optimization of drug delivery.

AN MRI-compatible&#160;custom-designed laser-based heating apparatus has been developed to provide local heating of subcutaneous tumors in order to activate release of agents from thermosensitive liposomes specifically at the tumor region.

定制设计的基于激光的加热装置顺铂触发释放的热敏脂质体与磁共振成像制导

Liposomes have been employed as drug delivery systems to target solid tumors through exploitation of the enhanced permeability and retention (EPR) effect ...

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

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

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

高通量单细胞的分离和文化的微流体平台

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

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

活微生物组共培养与人的微工程肠绒毛在肠道上一个芯片微流体装置

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

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 ...