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。成纤维细胞的血管长度成反比, 成纤维间的距离为31, 因此较短的距离有利于稳定的血管形成。然而, 要打开球体孔 (直径1毫米) 而不破坏通道1和 3, 通道2的最小宽度为1.5 毫米。
虽然球体位于井底, 但血管根部偶尔与 microposts 或微断开连接 (图 5)。在这种情况下, 没有试剂可以达到血管腔通过微 (通道1或 3)。虽然我们不能完全解决这个问题, 我们推测问题是由于纤维蛋白凝胶表面的血管内皮细胞数量不足 (步骤 4.2. 1-4. 2.6)。确保血管内皮细胞稳定地连接2通道的侧壁。
为了成功地将球体注入井中, 在步进4.1.7 中优化微尖的内径和外径是很重要的: 1) 内径应大于球体的内径。2) 外直径应适合井边, 使凝胶在注射过程中不会发生在油井顶部, 并且在凝胶注射后可以很容易地去除尖端。在目前的协议 (φ1毫米的球体井), 约700µm 是最大直径为可注射球体。如果井径设计得比本协议大, 则可以注入较大的球体, 因为尖端可以被切割成有较大的内径。通过改变在96井板中收获的细胞数, 可以很容易地控制球体直径。
该协议首先展示了一种新的微流控平台在球体中构造 perfusable 血管网络。尽管与血管内皮细胞的共培养允许在球状18、23、24 中形成类似于容器的结构, 但由于腔的死端, 这些不 perfusable。因为成纤维细胞存在于多种组织中 (骨骼、脂肪和肿瘤,等等), 通过增加一些其他靶向细胞到共培养球体, 可以预期各种球体的血管化, 这可以模仿在体内的环境比传统的球形培养更好。为了扩大该技术的应用, 今后的工作将包括球体的血管化, 而不添加成纤维细胞。最近的一些工作报告骨髓基质细胞32和肌肉细胞33可以诱导微流控装置的血管生成。利用基质或肌肉细胞代替成纤维细胞, 可以增加微流控装置中可血管化的组织数量。该协议为组织血管化提供了一个基础平台, 是三维文化领域中期待已久的技术。

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><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>Surface profiler</td><td>Veeco</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)&amp;nbsp;</td><td>Kai Industries</td><td>BP-10F</td><td></td></tr><tr><td>Biopsy Punch (2.0mm)&amp;nbsp;</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&amp;bull; 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&amp;nbsp; 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>CO&lt;sub&gt;2&lt;/sub&gt; 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。
图 7a显示单元格播种后微流控设备的中心。明亮的领域和荧光图像在0天显示, 纤维蛋白凝胶只填充通道2没有任何渗漏到通道1和 3, 和血管内皮细胞成功地连接到侧壁的纤维蛋白凝胶。明亮的场图像集中在被装载的球体和 microposts, 表明球体正确地在设备的底部被安定了。1天观察到血管生成苗, 芽长随时间增加 (图 7b)。在3天, 最长的芽到达球体和7天, 大多数的血管生成苗到达球体 (平均距离从通道1和3到球体 < 500 µm)。在最好的情况下, 在设备培养4天后, 可以观察到血管流明的流动。血管的角度被定义为血管尖端的方向和球体的中心从血管根。图 7c显示设备区域性7天内量化的维度角度。血管的角度随着时间的推移而减少, 表明血管生成芽向球体迁移。
图 8指示血管内皮细胞和 GFP-血管内皮细胞合并的维管束网络部分。RFP-血管内皮细胞和 GFP-血管内皮细胞协调形成一个单一的血管腔, 如箭头头显示, 这清楚地表明从1和3通道的血管生成芽与 RFP 血管内皮细胞在球体和形成一个连续的血管网络。为确定血管网的 perfusability, FITC-葡聚糖注入1通道。FITC-葡聚糖在通道1流入构造的血管网络和球体内部, 最后到达通道 3 (图 9)。在 FITC 葡聚糖溶液的灌注过程中, 血管网络没有渗漏到外空间。此前已表明, 在血管腔内注入的小分子通过血管壁, 并与外地区的细胞发生反应。此外, 该血管网络的渗透系数显示接近于该体内27。这些结果表明, 综合血管网可以为球体提供养分, 去除废弃物。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57242/57242fig1.jpg"> 图 1: 设计和微流控设备的照片.(a) 微流控装置的设计概述。灰色区域表示三微流控通道由梯形 microposts 分离。2频道的球体文化很好。右图显示了左边红色矩形中放大的视图。(b) 以红色墨水填充其通道的微流控装置的照片。<a href="/files/ftp_upload/57242/57242fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57242/57242fig2.jpg"> 图 2: 实验时间.<a href="/files/ftp_upload/57242/57242fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57242/57242fig3v2.jpg"> 图 3: 将球体加载到微流控设备中的方法.(a) 在两个不同的盘子里, 用球体制作一滴 MS 和媒体。然后, 用凝血酶将球体转移到 MS 中。(b) 在球体射入过程中, 装置的示意图剖面图。过量的凝胶流过孔2A 和2B。然而, 由于物理约束, 球体停留在设备的底部。(c) 设备在孵化器中的示意图。湿 Kimwipe 放在一个100毫米的盘子里, 两个35毫米的盘子里放上了 Kimwipe。<a href="/files/ftp_upload/57242/57242fig3v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57242/57242fig4.jpg"> 图 4: 用于将单元格播种到设备上的吸管提示的照片.左边白色提示是为孔 1A, 1B, 3A 并且3C 并且转移球形到一滴纤维蛋白凝胶 (步 4.1.9, 4.1.13, 4.2.4 并且 4.2.6)。中间黄色提示是为球形的汇集从96井 (步 4.1.7)。右白尖是为球体井 (步 4.1.11)。切口前的提示也显示在照片中。<a href="/files/ftp_upload/57242/57242fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57242/57242fig5.jpg"> 图 5: 非 perfusable 血管.血管生成苗从微没有连接到开放之间的 microposts 和失去连接之间的流明和微 (黑色箭头)。这是由1和3通道中的血管内皮细胞不足引起的。红色: 血管内皮细胞在球体, 绿色: GFP-血管内皮细胞从微。细胞在该装置中培养了14天。<a href="/files/ftp_upload/57242/57242fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57242/57242fig6v2.jpg"> 图 6: 定义血管根、尖端和球体的中心.(a) 确定血管根和尖端位置的方法来测量发芽长度。在对定时荧光图像进行校准后, 在0天的图像中减去在设备中进行培养几天后所拍摄的荧光图像。我们用 ImageJ 软件测定了减法后的芽长。(b) 球体中心的定义。血管内皮细胞的荧光图像由 ImageJ 软件二值化和测量。我们将质心定义为球体的中心。<a href="/files/ftp_upload/57242/57242fig6v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/57242/57242fig7.jpg"> 图 7: 在微流控装置中形成一个血管网络.(a) 在细胞播种后微流控装置中心的时间推移图像。红色: RFP-血管内皮细胞培养在球体, 绿色: GFP-血管内皮细胞在通道1和3收获。对芽长 (b) 和血管角 (c) (n = 42 芽) 的定量分析在3个设备中, 误差条指示标准错误 (东南亚))。发芽长度被定义为距离从血管内皮细胞的位置在天0到血管尖端 (b, 顶部)。角度 (∠TRS) 是由血管根 (R), 尖端 (T) 和球体 (c, 顶部) 的中心定义的。有关血管根、尖端和球体中心的定义, 请参见图 6 。这些数据是使用 ImageJ 软件获得的。对发芽长度和血管角度的定义进行了解释, 从梨本, Y. 等. 27.<a href="/files/ftp_upload/57242/57242fig7large.jpg" target="_blank">请单击此处查看此图的更大版本.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/57242/57242fig8v2.jpg"> 图 8: 由微和球体血管内皮细胞形成血管腔。(a) 在设备培养14天后对样品进行概述的荧光图像。黄色矩形表示 (b) 中显示的光学截面的 x y 位置。(b) 构建的血管网的 x y、y z 和 x z 光学截面。白色箭头表示血管腔。红色: 血管内皮细胞培养在球状, 绿色: GFP-血管内皮细胞培养在微, 蓝色: 细胞细胞核。(a) 不显示蓝色荧光, 因为 (a) 是固定前的图像。<a href="/files/ftp_upload/57242/57242fig8v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/57242/57242fig9.jpg"> 图 9: 使用构造的血管网络对球体进行灌注.明亮的领域 (a) 和荧光 (b) 球体的图像在7天以后在设备文化中。(c) FITC-葡聚糖 (70 kDa) 加载后同一球体的荧光图像。红色: 血管内皮细胞在球体和通道1和 3, 绿色: FITC-葡聚糖, 蓝色: 细胞细胞核。<a href="/files/ftp_upload/57242/57242fig9large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
辅助文件 1: 微流控设备的设计.该文件以 dxf 格式。<a href="/files/ftp_upload/57242/Supplementary_File_1.dxf" target="_blank">请单击此处下载此文件.</a>

Watch this Scientific Journal Video about Perfusable Vascular Network with a Tissue Model in a Microfluidic Device at JoVE.com

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

Universidad San Sebastian

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

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

内皮化微流体研究在血液病的微血管相互作用

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

科研

生物工程

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 ...

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 ...

制备基于微流体的血管化人类微肿瘤模型

Establishing a Physiologic Human Vascularized Micro-Tumor Model for Cancer Research

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical cancer research and therapeutic development.&#160;To overcome this problem, we have developed the vascularized microtumor (VMT), or tumor chip, a microphysiological system that realistically models the complex human tumor microenvironment. The VMT forms de novo within a microfluidic platform by co-culture of multiple human cell types under dynamic, physiological flow conditions. This tissue-engineered micro-tumor construct incorporates a living perfused vascular network that supports the growing tumor mass just as newly formed vessels do in vivo. Importantly, drugs and immune cells must cross the endothelial layer to reach the tumor, modeling in vivo physiological barriers to therapeutic delivery and efficacy. Since the VMT platform is optically transparent, high-resolution imaging of dynamic processes such as immune cell extravasation and metastasis can be achieved with direct visualization of fluorescently labeled cells within the tissue. Further, the VMT retains in vivo tumor heterogeneity, gene expression signatures, and drug responses. Virtually any tumor type can be adapted to the platform, and primary cells from fresh surgical tissues grow and respond to drug treatment in the VMT, paving the way toward truly personalized medicine. Here, the methods for establishing the VMT and utilizing it for oncology research are outlined. This innovative approach opens new possibilities for studying tumors and drug responses, providing researchers with a powerful tool to advance cancer research.

作者聚焦：创建人类血管化微肿瘤作为转化癌症研究的模型 

This protocol presents a physiologically relevant tumor-on-a-chip model to perform high-throughput basic and translational human cancer research, advancing drug screening, disease modeling, and personalized medicine approaches with a description of loading, maintenance, and evaluation procedures.

建立用于癌症研究的生理性人血管化微肿瘤模型

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical ...

癌症研究

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 ...

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 ...

Microfluidic Model to Mimic Initial Event of Neovascularization

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the initial stage varies dramatically from the following process of neovascularization. Although there are plenty of models to simulate different stages of neovascularization, an in vitro 3D model that capitulates the initial process of neovascularization under the corresponding stimulations of normal vasculature microenvironments is still lacking. Here, we reconstructed an in vitro 3D model that mimics the initial event of neovascularization (MIEN). The MIEN model contains a microfluidic sprouting chip and an automatic control, highly efficient circulation system. A functional, perfusable microchannel coated with endothelium was formed and the process of sprouting was simulated in the microfluidic sprouting chip. The initially physiological microenvironment of neovascularization was recapitulated with the microfluidic control system, by which ECs would be exposed to high luminal shear stress, physiological transendothelial flow, and various vascular endothelial growth factor (VEGF) distributions simultaneously. The MIEN model can be readily applied to the study of neovascularization mechanism and holds a potential promise as a low-cost platform for drug screening and toxicology applications.

Here, we provide a microfluidic chip and an automatically controlled, highly efficient circulation microfluidic system that recapitulates the initial microenvironment of neovascularization, allowing endothelial cells (ECs) to be stimulated by high luminal shear stress, physiological level of transendothelial flow, and various vascular endothelial growth factor (VEGF) distribution simultaneously.

模仿神经血管化初始事件的微流体模型

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the ...

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 ...

微流控装置中组织模型的 Perfusable 血管网

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内皮化微流体研究在血液病的微血管相互作用

微京尼平沉积技术的微模式化血管肌肉薄膜的扩展文化

缩微和3D微血管大会

建立用于癌症研究的生理性人血管化微肿瘤模型

建立用于癌症研究的生理性人血管化微肿瘤模型

建立用于癌症研究的生理性人血管化微肿瘤模型

影像引导下，血管源性微流控网络的基于激光的研制

微流控生物工程血管化组织和细胞器解体

模仿神经血管化初始事件的微流体模型

在微流体平台内开发 3D 组织人类心脏组织