这项研究由NIH 1R01HL140239-01向AMC资助。我们要感谢德雷塞尔大学的细胞成像中心。

1. 乳房上皮细胞生长和检测介质
<ol><li>MCF10A 乳房上皮细胞 注:非肿瘤不朽的乳房上皮细胞系来自纤维囊病患者43。细胞不表达雌激素受体。<ol>
<li>要准备 20μg/mL 表皮生长因子 （EGF），在 500 μL 的无菌 dH2O 中溶解 100μg 的亲皮 EGF，使 200 μg/mL EGF。在 dH 2 O 中将 200 微克/mL EGF 的 500 微升添加到 4.5 mL 的无菌0.1%BSA 中，以制作 20μg/mL EGF 库存解决方案。商店准备20μg/mL EGF在-20°C的别名长达12个月。</li>
		<li>要准备 500μg/mL 氢皮质松，在 1 mL 绝对乙醇中稀释 1 毫克氢皮质松（200 证明）。将 1 毫升无菌 DMEM F:12 添加到此混合物中，制成 500μg/mL 氢皮质松库存溶液。将氢皮质松库存报价存储在-80°C，最长达12个月。</li>
		<li>要准备 10μg/mL 霍乱毒素，在 1 mL 无菌 dH2O 中溶解 1 毫克霍乱毒素亲血粉，以制作 1 毫克/毫升霍乱毒素储存溶液。将霍乱毒素储存在 -80 °C 下长达 12 个月。</li>
		<li>准备增长介质， 添加 500 μL 的 EGF （20μg/mL）， 500μL 的水皮质松 （500μg/mL）， 5 μL 霍乱毒素 （1 毫克/mL）， 500 μL 牛胰岛素 （10 毫克/mL）， 10 mL 青霉素和链霉素， 和25毫升的马血清到500毫升的DMEM F:12的最终浓度为20 ng/mL EGF， 500克/mL氢皮质松，10克/mL霍乱毒素， 10 ng/mL 牛胰岛素，2% v/v 青霉素和链霉素，5% v/v 马血清。抗生素浓度增加到2%，以减少生物打印过程中的不育性:然而，抗生素浓度可以降低到1%。</li>
		<li>要准备检测介质，添加 500 μL 的水皮质松 （500μg/mL），5 μL 霍乱毒素 （1 毫克/mL）， 500μL 牛胰岛素 （10 毫克/mL）， 10 mL 青霉素和链霉素， 和25毫升的马血清到500毫升的DMEM F:12的最终浓度为500克/毫升氢皮质松，10克/毫升霍乱毒素，10克/毫升牛胰岛素，2%v/v青霉素和链霉素，和5%v/v马血清。</li>
	</ol>
</li>
	<li>MDA-MB-231 注:三阴性（缺乏雌激素受体、黄体酮受体和表皮生长因子受体2）乳腺癌上皮细胞系是由转移性乳腺癌44的女性患者的胸膜发泡产生的。细胞代表一种侵略性、侵入性且分化不良的乳腺癌。<ol>
<li>准备生长和检测介质， 添加50 mL的胎儿牛血清，10mL的青霉素和链霉素，和25mL的马血清到500mL的DMEM为最终浓度10%v/v胎儿牛血清，2%v/v青霉素和链霉素，和5%v/v马血清。</li>
	</ol>
</li>
</ol>2. 乳房上皮细胞培养
<ol><li>种子 500，000 MCF10A 或 MDA-MB-231 细胞在 10 厘米 （P100） 组织培养皿与 10 mL 的 MCF10A 或 MDA-MB-231 生长介质。MCF10A 和 MDA-MB-231 细胞在 48 小时内达到 +90% 的汇合。</li>
	<li>要 通过乳房上皮细胞， 首先用10 mL的温暖PBS清洗细胞。</li>
	<li>通过在菜中加入 2 mL 的 0.05% 特里普辛 - EDTA 来分离乳房上皮细胞。将菜放入 37 °C、5% CO2 孵化器中 20-25 分钟。</li>
	<li>将5 mL的MCF10A或MDA-MB-231生长介质添加到胰腺素细胞中和胰腺素。上上下下将细胞介质混合物向上和向下移到悬生细胞并分解细胞簇。</li>
	<li>将电池悬架添加到 15 mL 圆锥管和离心机中，1，200 x g， 持续 3 分钟。小心地吸气超纳坦，并在 MCF10A 或 MDA-MB-231 生长介质的 5 mL 中重新悬浮细胞颗粒。</li>
	<li>板 1 mL 的细胞悬浮在 5 个新的 10 厘米组织培养皿以及 MCF10A 或 MDA-MB-231 生长介质。将菜肴放在 37 °C、5% CO2 孵化器中。细胞将在2-3天内再次通过。MCF10A细胞可以维持到35号通道，之后它们显示形态变化。MDA-MB-231细胞可以维持到24号通道，之后它们显示出形态的变化。</li>
</ol>3. 乳房上皮球形形成
<ol><li>在开始检测前冻结 200 μL 移液器提示 30 分钟。在整个过程中将生长因子减少矩阵溶液（例如矩阵）（10 毫克/mL）保留在冰上。</li>
	<li>使用冰冷 200μL 移液器提示将 30 μL 冰冷矩阵溶液缓慢移液到 8 井室滑梯的每口井中。从两侧开始，沿着拐角拖动移液器尖端，在油井中央添加最后一滴水，以确保每个油井均匀涂层。在此步骤中避免气泡，以确保均匀的矩阵溶液层。为每口井使用新的移液器提示。</li>
	<li>孵化室在 37 °C 中滑动，5% CO2 孵化器 15-20 分钟，以聚合矩阵溶液。</li>
	<li>MCF10A检测介质中悬垂的尝试性MCF10A细胞在200，000个细胞/mL或在MDA-MB-231生长介质中以200，000个细胞/mL重新悬垂的尝试性MDA-MB-231细胞。</li>
	<li>如果使用 MCF10A 细胞，则通过在 5 mL 的检测介质中添加 5 μL 的 EGF （20 μg/mL） 和 100 μL 矩阵溶液来准备新鲜的 MCF10A 球形生长介质，最终浓度为 2% 矩阵溶液。</li>
	<li>从孵化器中取出预涂有矩阵溶液的腔室滑梯。在每个井中添加 50 μL 的 MCF10A 或 MDA-MB-231 细胞悬架（10，000 个细胞）。如果使用 MCF10A 细胞，添加 450μL 的 MCF10A 球体生长介质。如果使用 MDA-MB-231 细胞，添加 450μL 的 MDA-MB-231 生长介质预混合 2% 矩阵溶液 （9 μL）。立即将腔室滑动放置在 37 °C 中，5% CO2 孵化器中。</li>
	<li>每4天更换一次介质。使用 200μL 移液器尖端从每口井的一角小心地移出 200μL 旧介质。在此过程中，将腔室滑动倾斜至 45°，以确保球体不受干扰。添加 200 μL 的新鲜制备的 MCF10A 球形生长介质或 MDA-MB-231 生长介质，以前与 2% 矩阵溶液混合到每个油井的拐角处，以确保球体保持附着在矩阵层上。仅更换了约 50% 的介质，以便球形产生的所有细胞因子不会完全耗尽。 注:MCF10A乳房上皮球体需要长达8天的时间两极分化并形成空心中心。MDA-MB-231乳房上皮球体需要长达5天的时间才能形成，并且没有空心中心。</li>
</ol>4. 内皮细胞网络形成
<ol><li>人类脐带静脉内皮细胞（HUVEC）培养<ol>
<li>添加含有胰岛素生长因子、成纤维细胞生长因子、血管内皮生长因子、抗坏血酸、表皮生长因子的单一内皮生长中-2套件的内容， 肝素和根霉素-安非他明B，以及50mL的胎儿牛血清，5 mL青霉素-链霉素和5 mL的200m谷氨酰胺到500mL的内皮基础中等2（EBM-2），以创建完整的内皮生长介质（EGM-2）。</li>
		<li>在 EGM-2 的 10 mL 中，在 10 厘米组织培养皿中播种 500，000 个内皮细胞。将细胞放置在 37 °C、5% CO2 孵化器中，直到它们达到 +80% 的汇合度（约 48 小时）。</li>
		<li>通过细胞，用10mL的温暖PBS清洗内皮细胞。通过在 10 厘米组织培养皿中添加 2 mL 的 0.05% 特里普辛 - EDTA 来分离 Huvec 。按相位对比显微镜密切监测细胞。细胞准备好时，他们被球起来，但保持连接到菜。</li>
		<li>小心地从盘子里吸出试食剂。在菜中加入 8 mL 的 EGM-2。通过在整个菜面上上下管道 EGM-2 来洗净盘子中的细胞。</li>
		<li>或者，在试金石细胞中加入5 mL的EGM-2，以中和试金石。上上下下将细胞介质混合物向上和向下移到悬生细胞并分解细胞簇。将电池悬架添加到 15 mL 圆锥管和离心机中，1，200 x g， 持续 3 分钟。小心地吸食超纳坦，并在 10 mL 的 EGM-2 中重新悬浮细胞颗粒。</li>
		<li>在 10 厘米组织培养盘中将 1 mL 的细胞悬浮液添加到 9 mL 的 EGM-2 中。将菜肴放在 37 °C、5% CO2 孵化器中。每2天交换2/3的介质与新鲜的EGM-2。细胞将在3-5天内通过。HUVEC 可以保持到第 8 段，之后它们无法形成网络。</li>
	</ol>
</li>
	<li>内皮细胞（HUVEC）网络形成</li>
	<li>在开始检测前冻结 200 μL 移液器提示 30 分钟。在整个过程中将生长因子减少矩阵溶液（10 毫克/mL）保留在冰上。</li>
	<li>使用冰冷 200μL 移液器提示将 30 μL 冰冷矩阵溶液缓慢移液到 8 井室滑梯的每口井中。从两侧开始，沿着拐角拖动移液器尖端，在油井中央添加最后一滴水，以确保每个油井均匀涂层。在此步骤中避免气泡，以确保均匀的矩阵溶液层。为每口井使用新的移液器提示。</li>
	<li>孵化室在37°C、5%CO2 孵化器中滑动15-20分钟，以聚合马特里格尔。<ol>
<li>在 37 °C、5% CO2 孵化器中，用 10 μL 的红细胞跟踪器 （1:1000） 预染色 10 厘米的 HUVEC 盘 30 分钟。</li>
		<li>用10mL的温暖PBS清洗内皮细胞。通过在 10 厘米组织培养皿中添加 2 mL 的 0.05% 特里普辛 - EDTA 来分离 Huvec 。将菜放入 37 °C、5% CO2 孵化器中 5 分钟，以完全分离细胞。</li>
		<li>将 5 mL 的 EGM-2 添加到菜中以中和试金石。将细胞悬架转移到 25 mL 圆锥管中。离心机 HUVEC 在 1，200 x g 5 分钟。吸气超纳坦，并在5mL的无血清EBM-2中重新悬浮细胞颗粒。</li>
		<li>使用Trypan蓝色计数细胞以确定可行的细胞。创建 1 x 106 单元格/mL 解决方案。</li>
		<li>使用 200μL 的无血清 EBM-2 将 100，000 个 HUVEC 添加到每个油井中。如果添加更多的细胞，他们将创建一个表面单层，而不是一个管网络。</li>
		<li>在37°C、5%CO2 孵化器中孵育HUVEC。6 小时后，按相对比显微镜对图像 HUVEC 网络。网络现在已准备好用乳房球形进行培养。</li>
	</ol>
</li>
</ol>5. 预形成的HUVEC网络上的生物打印乳房上皮球体
注:双喷嘴生物沉积系统应用于生物制剂过程。在这种情况下，该系统有三个运动臂，允许微米规模的空间控制材料沉积，以及两个螺丝驱动电机沉积生物墨水从10 mL注射器。该系统应具有高效颗粒空气过滤系统以及紫外线灭菌功能，以在生物打印过程中保持无菌环境。生物打印机在打印过程前进行一小时的紫外线消毒。
<ol><li>如前所述，创建乳房上皮球体和 HUVEC 网络。通过在代表性相对比显微镜图像中数球体来估计每口井中乳房上皮球体的数量。</li>
	<li>从 1000μL 移液器尖端切下 0.5 厘米。使用切割移液器尖端小心地将所有球形从 8 井室滑梯中移出并放入 50 mL 管中。在选定的生物墨水中以 100 个球形/100 μL 重新悬生球体。 注:较高的球形浓度可能导致球形聚类，并防止球形和内皮网络之间的交互可视化。</li>
	<li>通过 70μm 细胞过滤器传递球形混合物，以去除任何大型或聚类球形。</li>
	<li>将池状球体加载到 10 mL 无菌注射器中，并用 25 光量无菌针盖上。将注射器连接到生物沉积系统。</li>
	<li>在 8 井室幻灯片中吸气 HUVEC 网络中的介质。</li>
	<li>以 1 mL/min 的流速将 100μL 的乳房上皮球体挤入 HUVEC 网络的 6 口井中。作为控件维护 2 口 HUVEC 网络井。</li>
	<li>如果使用 HUVEC 网络上打印的 MCF10A 球形生长介质添加 400 μL，或添加 400 μL MDA-MB-231 增长介质，如果使用 HUVEC 网络上打印的 MDA-MB-231 球形，则添加以前与 2% 矩阵解决方案混合的生长介质。应在 HUVEC 控制井中使用相应的球形生长介质。</li>
	<li>在37°C、5%CO2 孵化器中孵育共同培养物24-96小时，无需更换媒体。共同文化将在原来的媒介中保持生存长达四天。</li>
</ol>6. 康福科显微镜
<ol><li>免疫荧光和PBS-甘氨酸洗涤缓冲准备<ol>
<li>通过添加 2.5 克阿齐德钠、5 克牛血清白蛋白、10 mL 的 Triton X-100 和 2.05 mL 的 Tween-20，在 10 倍 PBS 中加入 2.5 克 Azide 钠，从而准备免疫荧光 （IF） 缓冲 10 倍库存溶液。将pH调整为7.4。将库存溶液在 4 °C 下存储长达一年，以避免沉积。</li>
		<li>通过在 450 mL 的无菌去电化水中稀释 50 mL 的 10 倍 IF 缓冲器创建一个工作 IF 缓冲区。将工作解决方案在室温下存储长达一周。</li>
		<li>通过将 37.5 克甘氨酸添加到 10 倍 PBS 的 500 mL 中，准备 10 倍 PBS-甘氨酸缓冲液库存解决方案。将pH调整为7.4。将库存溶液在 4 °C 下存储长达 6 个月，以避免沉积。</li>
		<li>通过在 450 mL 的无菌去电化水中稀释 50 mL 的 10 倍 PBS-甘氨酸缓冲液，创建有效的 PBS-甘氨酸溶液。将工作解决方案存储在 4 °C 下长达一周。</li>
	</ol>
</li>
	<li>标签生物打印样品和图像由康福科显微镜<ol>
<li>从生物打印的3D共同培养和冲洗3次与温暖的PBS吸气介质。修复生物打印的3D共培养物与4%的副甲醛1小时在室温下。用 1x PBS-甘氨酸冲洗样品 3 次，20 分钟。</li>
		<li>用IF缓冲器将样品与10%山羊血清混合90分钟（主块），然后用IF缓冲区加10%山羊血清和Affinipure Fab片段（1:100，次要块）块样品。</li>
		<li>如果使用 MCF10A 球体，则在 4 °C 的二次阻塞缓冲区中标记具有集成蛋白 ® 6 （1:100） 的原发抗体的样品，然后是 Alexa Fluor 488 次要抗体 （1:200） 和 Hoescht 33342 （1:1000）， 在室温下防止光线照射 1 小时。MCF10A细胞系表示高水平的集成+6，这是显示球体极化和形态学的关键。</li>
		<li>如果使用MDA-MB-231球形，表示低浓度的集成+6，则用亚历克萨氟488 phalloidin（1:100）和Hoescht 33342（1:1000）在室温下在隔光下标记样品4小时。Phalloidin 支持作用灯丝可视化，因此可以评估球形无定形和侵入性形态。</li>
		<li>用1X PBS-甘氨酸清洗样品3次，20分钟。</li>
		<li>使用制造商的工具移除机舱滑梯，准备安装样品。给每口井添加一小滴防脂溶液。在每个房间滑梯上放置一个 22 mm x 60 mm 的盖片，并小心地用透明指甲油密封边缘。</li>
		<li>使用共分显微镜作为 Z 堆栈的图像样本在 5 μm 步骤中将 +10 切片堆叠在一起。如果需要，使用细胞成像软件中的扩展对焦命令将 Z 平面压缩到单平面上。</li>
		<li>使用图像 J 将球形粘附量化到内皮网络。可使用具有适当粒子大小和圆形的分析插件量化粘附球体的数量。将附加的球形数标准化到图像区域。</li>
		<li>为了确保可重复性，量化共同培养的 4 x 4 瓷砖图像中粘附球体的数量。如果球形数在统计上明显低于其他实验，则应重复该实验。</li>
	</ol>
</li>
</ol>

作者没有什么可透露的。

此协议是首次在其 3D 架构中与内皮细胞在其 3D 架构中共同培养的生物打印球体。关键协议步骤包括乳房上皮球体和HUVEC网络的初始形成。在喂养乳房上皮球体时必须格外小心，因为它们很容易被矩阵溶液打乱。同样，乳房上皮球体在从矩阵溶液上移开并混合到网络中时，必须小心对待。HUVEC 网络不应以过高的密度进行镀层，也不应离开超过 16 小时，因为它们将分别形成单层或死亡。最后，所有?...

3D体外血管化肿瘤模型是癌症生长和转移的机械研究的重要工具。特别是对于乳腺癌，在Matrigel培养的乳房上皮细胞组织成极化球形，更类似于体内乳腺癌结构1，2，3，4，5，6，7，8。3D乳房上皮细胞培养也影响细胞功能，3D培养物在表皮生长因子（EGF）受体调节8、9方面表现出差异:电基因功能，包括 ErbB210:生长和凋亡信号11，12:和化疗耐药性13，14。血管内皮细胞在3D与传统2D文化15、16、17、18中对环境刺激的反应相似。然而，对血管内皮和乳房上皮相互作用的理解大部分来自使用条件介质或Transwell插入物的2D培养，或3D模型，其中两种细胞类型物理分离19，20，21，22，23。这些共同培养模型提供了有限的生理洞察力，因为3D培养和细胞接触对血管内皮-乳房上皮细胞相互作用24，25，26至关重要。
3D癌症模型是使用各种技术制造的，包括悬挂下降球形形成，生物打印，磁性组装，培养在水凝胶或工程脚手架5，27，28，29。最近，3D肿瘤模型被创建，在各自的3D结构中排列了多种细胞类型。在片上肿瘤平台的一个例子中，癌症、内皮细胞和频闪细胞被混合到基质中，然后注射到聚二甲基硅氧烷（PDMS）装置中的三个中央组织室中。组织室由代表动脉和静脉的两个外通道包围。经过5-7天的培养，内皮细胞形成微血管网络，癌细胞增殖，在血管附近形成小肿瘤。然后，该平台用于筛选药物和药物组合30。其他肿瘤片上平台已经创建，以研究转移和癌症类型与特定的机械刺激（例如，在肺部的机械应变）31，32。然而，这些平台通常不包括血管和癌症在其各自的3D结构。
生物合成在推进体外血管化肿瘤模型方面显示出巨大的希望，因为它能够对细胞位置进行严格的空间控制。尽管生物印刷在过去十年中增长，但很少有研究特别侧重于肿瘤33，34。在一个例子中，用明胶/藻酸盐/纤维蛋白原水凝胶对HiLa细胞进行3D打印，以创建体外宫颈癌模型。肿瘤细胞被生物打印为单个细胞，然后允许形成球形，这表明更高的增殖率，增加矩阵金属蛋白酶表达，和更高的化学耐附性比细胞在2D培养35。在这些研究中，像许多其他36，37，分离细胞悬浮物被生物打印，然后细胞培养物被提供所需的机械和生化线索，使细胞形成一个3D结构。然而，细胞自组装可能需要数天或数周的时间，可能需要复杂的空间和时间环境提示，或在两种细胞类型共同培养时可能不会发生。例如，乳腺上皮细胞在2D共生中诱发内皮细胞死亡，分离的乳房上皮细胞在生物印在藻酸盐/明胶水凝胶38中时没有形成3D球形。分离的乳腺上皮或癌细胞只有在包裹在圆形 PDMS 模具中时，才会在藻酸盐生物沉积中形成球形。在其他情况下，球体是使用悬浮液滴在超低附件圆形井板中形成的，然后混合成藻酸盐基生物墨水39，40。
我们现在在此协议中描述了一种替代的3D组织生物制造方法。我们描述的不是种子分离细胞和等待这些细胞形成3D结构，而是描述如何在血管管网络上创建和生物打印3D肿瘤球体，以创建一个肿瘤共生模型，几乎可以立即使用。肿瘤球体可以在体外生长或从人体组织（器官）中提取。同样，血管管可以生长，也可以从脂肪组织微血管片段中提取。生物素的范围从生物不活跃的藻酸盐到高度生物活性的马特里格尔41。由于这种3D肿瘤共生模型可以创建与各种细胞结构和生物素，它可以结合多种细胞类型，细胞外矩阵，和化学基质梯度15，42。虽然在其目前的配方中，内皮网络无法注入，但未来的迭代可以将此方法与微流体或片上系统集成。将3D乳房上皮球体打印到内皮网络上，使人类乳房模型能够快速生物合成，用于药物测试和个性化精密医学27。

<table><tbody><tr><td>37&amp;deg;C incubator, 5% CO2 and 95% humidity</td><td>Sanyo</td><td>MCO-20AIC</td><td>Cell incubation</td></tr><tr><td>3D Bio printer</td><td>custom-made</td><td>None</td><td>Used for bioprinting</td></tr><tr><td>8-well chamber slides</td><td>VWR, Radnor, PA</td><td>53106-306</td><td>for seeding spheroids</td></tr><tr><td>25-gauge needle</td><td>Sigma, St. Louis, MO</td><td>Z192406-100EA</td><td>bioprinting syringe needle</td></tr><tr><td>Absolute ethanol (200 proof )</td><td>Sigma, St.Louis, MO</td><td>E7023-500ML</td><td>reconsitution of media components</td></tr><tr><td>Affinipure F(ab&amp;prime;)2 fragment goat anti-mouse IgG</td><td>Jackson ImmunoResearch, West Grove, PA</td><td>115006020</td><td>secondary block - Immunofluorescence</td></tr><tr><td>Alexa Fluor 488 (1:200)</td><td>Thermo Fisher, Waltham, MA</td><td>A-11006</td><td>Seconday antibody-Immunofluorescence</td></tr><tr><td>Bovine insulin</td><td>Sigma, St.Louis, MO</td><td>I-035-0.5ML</td><td>MCF10A Media additive</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma, St.Louis, MO</td><td>A2153-500G</td><td>Blocking agent -Immunofluorescence</td></tr><tr><td>Falcon 70 &amp;micro;m Cell Strainer</td><td>Corning, Corning, NY</td><td>352350</td><td>Remove large or clustered spheroids</td></tr><tr><td>CellTracker&amp;trade; Red CMTPX Dye</td><td>Thermo Fisher, Waltham, MA</td><td>C34552</td><td>pre-stain for HUVEC tubes</td></tr><tr><td>Compact Centrifuge</td><td>Hermle- Labnet, Edison ,NJ</td><td>Z206A</td><td>For cell centrifugations</td></tr><tr><td>Cholera Toxin</td><td>Sigma, St.Louis, MO</td><td>C8052-.5MG</td><td>MCF10A Media additive</td></tr><tr><td>Conical tubes 15 mL</td><td>VWR, Radnor, PA</td><td>62406-200</td><td>Collecting and resuspending cells</td></tr><tr><td>Countess II-FL Cell counter</td><td>Thermo Fisher, Waltham, MA</td><td>AMQAF1000</td><td>counting cells</td></tr><tr><td>Glass pipettes (10 mL)</td><td>VWR, Radnor, PA</td><td>76184-746</td><td>cell resuspension</td></tr><tr><td>DMEM F:12</td><td>Thermo Fisher, Waltham, MA</td><td>11320033</td><td>MCF10A basal media</td></tr><tr><td>DMEM 1X</td><td>VWR, Radnor, PA</td><td>10-014-CV</td><td>MDA-MB-231 basal media</td></tr><tr><td>Endothelial Basal Medium-2 (EBM-2)</td><td>Lonza, Durham, NC</td><td>CC-3156</td><td>HUVEC basal media</td></tr><tr><td>Endothelial Growth Medium-2 (EGM-2)</td><td>Lonza, Durham, NC</td><td>CC-3162</td><td>Accompanied with a Bulletkit (containing growth factors)</td></tr><tr><td>Alexa Fluor&amp;trade; 488 Phalloidin</td><td>Thermo Fisher, Waltham, MA</td><td></td><td>Labelling MDA-MB-231 spheroids</td></tr><tr><td>Fetal Bovine serum</td><td>Cytiva, Logan, UT</td><td>SH30071.03</td><td>HUVEC/MDA-MB-231 media additive</td></tr><tr><td>Goat serum</td><td>Thermo Fisher, Waltham, MA</td><td>16210064</td><td>Immunofluorescence labelling component</td></tr><tr><td>Glycine</td><td>Sigma, St.Louis, MO</td><td>G8898-500G</td><td>immunofluorescence buffer component</td></tr><tr><td>Hoescht 33342</td><td>Thermo Fisher, Waltham, MA</td><td>62249</td><td>Nuclei stain immunofluorescence</td></tr><tr><td>Horse Serum</td><td>Thermo Fisher, Waltham, MA</td><td>16050130</td><td>MCF10A Media additive</td></tr><tr><td>Hydrocortisone</td><td>Sigma, St.Louis, MO</td><td>H0888-5G</td><td>MCF10A Media additive</td></tr><tr><td>Human Umblical Vein Endothelial cells (HUVECs)</td><td>Cell applications, San Diego , CA</td><td>200-05f</td><td>Endothelial cell lines</td></tr><tr><td>Integrin &amp;alpha;6</td><td>Millipore, Billerica, MA</td><td>MAB1378</td><td>Immunofluorescence spheroid labelling component</td></tr><tr><td>Live Dead assay</td><td>Thermo Fisher, Waltham, MA</td><td>L3224</td><td>Live and dead cell stain assay for cell viability</td></tr><tr><td>LSM 700 Confocal microscope</td><td>Zeiss, Thornwood, NY</td><td></td><td>Used to visualize cells</td></tr><tr><td>Matrigel - growth factor reduced 10 mg/ml</td><td>VWR, Radnor, PA</td><td>354230</td><td>Spheroid formation</td></tr><tr><td>MCF10A cells</td><td>ATCC</td><td>CRL-10317</td><td>Breast cell line</td></tr><tr><td>MDA-MB-231 cells</td><td>ATCC</td><td>HTB-26</td><td>Breast cell line</td></tr><tr><td>Paraformaldehyde</td><td>Sigma, St.Louis, MO</td><td>158127-500G</td><td>cell fixative</td></tr><tr><td>Penicillin and streptomycin</td><td>Thermo Fisher, Waltham, MA</td><td>15140122</td><td>MCF10A / MDA-MB-231/HUVEC Media additive</td></tr><tr><td>Phosphate Buffered Saline 1X (PBS)</td><td>Thermo Fisher, Waltham, MA</td><td>7001106</td><td>Wash buffer for cells before trypsinization</td></tr><tr><td>Phosphate buffer saline 10X</td><td>Thermo Fisher, Waltham, MA</td><td>AM9625</td><td>immunofluorescence buffer component</td></tr><tr><td>Prolong gold antifade</td><td>Thermo Fisher, Waltham, MA</td><td>P36934</td><td>immunofluorescence mountant medium</td></tr><tr><td>Recombinant Human Epidermal Growth Factor, EGF</td><td>Peprotech, Rocky Hill, NJ</td><td>AF-100-15</td><td>MCF10A/ assay media component</td></tr><tr><td>Sodium Azide</td><td>Sigma, St.Louis, MO</td><td>S2002-25G</td><td>immunofluorescence buffer component</td></tr><tr><td>Sterile syringe (10 mL)</td><td>VWR, Radnor, PA</td><td>75846-757</td><td>bioprinting process</td></tr><tr><td>Tissue culture dish (10cm)</td><td>VWR, Radnor, PA</td><td>25382-166</td><td>monolayer cell culture</td></tr><tr><td>Triton X-100</td><td>Sigma, St.Louis, MO</td><td>T8787-250ML</td><td>immunofluorescence buffer component</td></tr><tr><td>Trypan blue 0.4%</td><td>Thermo Fisher, Waltham, MA</td><td>15250061</td><td>cell counter additive</td></tr><tr><td>Trypsin-EDTA 0.05%</td><td>Thermo Fisher, Waltham, MA</td><td>25300054</td><td>cell detachment</td></tr><tr><td>Tween -20</td><td>Thermo Fisher, Waltham, MA</td><td>85113</td><td>immunofluorescence buffer component</td></tr><tr><td>Vascular Endothelial Growth factor (VEGF&lt;sub&gt;165&lt;/sub&gt;)</td><td>Peprotech, Rocky Hill, NJ</td><td>100-20</td><td>HUVEC tube additive</td></tr><tr><td>Volocity 6.3 cell imaging software</td><td>PerkinElmer, Hopkinton, MA</td><td></td><td>Z stack compresser</td></tr></tbody></table>

direct bioprinting of 3d multicellular breast spheroids onto endothelial networks

生物打印正在成为制造3D人类癌症模型的有希望的工具，能够更好地回顾体内组织结构的关键特征。在目前的逐层挤压生物打印中，单个细胞与复杂的空间和时间线索一起挤出生物沉淀物，以促进分层组织自我组装。然而，这种生物制药技术依赖于细胞、生物素和生化和生物物理线索之间的复杂相互作用。因此，自组装可能需要数天甚至数周的时间，可能需要特定的生物墨水，并且当涉及多个细胞类型时，可能并不总是发生。因此，我们开发了一种技术，直接生物打印预先形成的3D乳房上皮球体在各种生物墨水。生物打印预形成的3D乳房上皮球体在打印后维持其生存能力和极化结构。此外，我们还将3D球体打印到血管内皮细胞网络上，以创建共培养模型。因此，新的生物打印技术迅速创造了一个与生理相关的3D人体乳房模型，其成本更低，灵活性也比传统生物打印技术高。这种多才多艺的生物打印技术可以推断出来，以创建其他组织的其他生物墨水的3D模型。

乳房上皮细胞应自我组织成3D球形后，5-8天的培养矩阵溶液和文化介质与2%的矩阵溶液。非肿瘤性MCF10A乳房上皮球体应呈圆形，并具有空心中心，与整数+6偏振到球体的外缘（图1，内嵌显示空心中心）。高度侵入性的MDA-MB-231乳腺癌上皮细胞形成不规则球形。当球形直径约为100-300μm时，应使用球形。当球体变得太大，并接近，球体将连接在一起，形成巨型球体。此外，MDA-MB-231...

Watch this Scientific Journal Video about Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks at JoVE.com

Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks

该协议的目标是将生物打印乳房上皮细胞作为多细胞球体直接印在预先形成的内皮网络上，以快速创建可用于药物筛选研究的3D乳腺内皮共生模型。

将3D多细胞乳房球体直接生物打印到内皮网络

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue ...

direct-bioprinting-3d-multicellular-breast-spheroids-onto-endothelial

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

4.8K 次观看.  Drexel University. 该协议的目标是将生物打印乳房上皮细胞作为多细胞球体直接印在预先形成的内皮网络上，以快速创建可用于药物筛选研究的3D乳腺内皮共生模型。

视频: Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved.
This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research.

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Crosstalk between mammary epithelial cells and endothelial cells importantly contributes to breast cancer progression, tumor growth, and metastasis. In this study, spheroids have been made from breast cancer cells together with vascular and/or lymphatic endothelial cells and demonstrate their applicability as an in vitro system for breast cancer research.

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能 体外 模型

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its ...

科研

癌症研究

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of three-dimensional bioprinting in tissue engineering has generated new methods for the printing of cells and matrix to fabricate biomimetic tissue constructs. The solid freeform fabrication (SFF) method developed for three-dimensional bioprinting uses an additive manufacturing approach by depositing droplets of cells and hydrogels in a layer-by-layer fashion. Bioprinting fabrication is dependent on the specific placement of biological materials into three-dimensional architectures, and the printed constructs should closely mimic the complex organization of cells and extracellular matrices in native tissue. This paper highlights the use of the Palmetto Printer, a Cartesian bioprinter, as well as the process of producing spatially organized, viable constructs while simultaneously allowing control of environmental factors. This methodology utilizes computer-aided design and computer-aided manufacturing to produce these specific and complex geometries. Finally, this approach allows for the reproducible production of fabricated constructs optimized by controllable printing parameters.

A Cartesian bioprinter was designed and fabricated to allow multi-material deposition in precise, reproducible geometries, while also allowing control of environmental factors. Utilizing the three-dimensional bioprinter, complex and viable constructs may be printed and easily reproduced.

Bioprinted蜂窝构造的可行性使用三分配器笛卡尔打印机

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of ...

生物工程

Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and fibroblasts are first isolated, counted and mixed at desired cell ratios. They are co-cultured in individual wells in ultra-low attachment 96-well plates. Within 3 days, beating spheroids form. These spheroids are then picked up by a nozzle using vacuum suction and assembled on a needle array using a 3D bioprinter. The spheroids are then allowed to fuse on the needle array. Three days after 3D bioprinting, the spheroids are removed as an intact patch, which is already spontaneously beating. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

使用3D生物印刷创建心脏组织展示球体的机械整合

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and ...

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 ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

微流控装置中组织模型的 Perfusable 血管网

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

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully recapitulate aspects of cancer progression such as proliferation and invasion. Although patient-derived organoids and patient-derived xenograft organoids are pathophysiologically relevant, they are costly to propagate, difficult to manipulate, and comprised primarily of the most proliferative cell types within the tumor microenvironment (TME). These limitations prevent their use for elucidating cellular mechanisms of disease progression that depend upon tumor-associated stromal cells which are found within the TME and known to contribute to metastasis and therapy resistance. 
Here, we report on methods for cultivating epithelial-stromal multicellular 3D cultures. The advantages of these methods include a cost-effective system for rapidly generating organoid-like 3D cultures within scaffold-free environments that can be used to track invasion at single-cell resolution within hydrogel scaffolds. Specifically, we demonstrate how to generate these heteromulticellular 3D cultures using BT-474 breast cancer cells in combination with fibroblasts (BJ-5ta), monocyte-like cells (THP-1), and/or endothelial cells (EA.hy926). Additionally, differential fluorescent labeling of cell populations enables time-lapse microscopy to define 3D culture assembly and invasion dynamics. 
Notably, the addition of any two stromal cell combinations to 3D cultures of BT-474 cells significantly reduces circularity of the 3D cultures, consistent with the presence of organoid-like or secondary spheroid structures. In tracker dye experiments, fibroblasts and endothelial cells co-localize in the peripheral organoid-like protrusions and are spatially segregated from the primary BT-474 spheroid. Finally, heteromulticellular 3D cultures of BT-474 cells have increased hydrogel invasion capacity. Since we observed these protrusive structures in heteromulticellular 3D cultures of both non-tumorigenic and tumorigenic breast epithelial cells, this work provides an efficient and reproducible method for generating organoid-like 3D cultures in a scaffold-free environment for subsequent analyses of phenotypes associated with solid tumor progression.

There is a critical need for 3D cancer models that capture heterocellular crosstalk to study cancer metastasis. Our study presents the generation of heteromulticellular stromal-epithelial in a scaffold and scaffold-free environment that can be used to study invasion and cellular spatial distributions.

上皮癌细胞无支架 3D 培养物中的异多细胞基质细胞驱动侵袭

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully ...

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell lines using conventional two-dimensional (2D) cell culture. These challenges can be overcome by using bioprinting techniques to build heterogeneous three-dimensional (3D) tumor models whereby different types of cells are embedded. Alginate and gelatin are two of the most common biomaterials employed in bioprinting due to their biocompatibility, biomimicry, and mechanical properties. By combining the two polymers, we achieved a bioprintable composite hydrogel with similarities to the microscopic architecture of a native tumor stroma. We studied the printability of the composite hydrogel via rheology and obtained the optimal printing window. Breast cancer cells and fibroblasts were embedded in the hydrogels and printed to form a 3D model mimicking the in vivo microenvironment. The bioprinted heterogeneous model achieves a high viability for long-term cell culture (&#62; 30 days) and promotes the self-assembly of breast cancer cells into multicellular tumor spheroids (MCTS). We observed the migration and interaction of the cancer-associated fibroblast cells (CAFs) with the MCTS in this model. By using bioprinted cell culture platforms as co-culture systems, it offers a unique tool to study the dependence of tumorigenesis on the stroma composition. This technique features a high-throughput, low cost, and high reproducibility, and it can also provide an alternative model to conventional cell monolayer cultures and animal tumor models to study cancer biology.

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

We developed a heterogeneous breast cancer model consisting of immortalized tumor and fibroblast cells embedded in a bioprintable alginate/gelatin bioink. The model recapitulates the in vivo tumor microenvironment and facilitates the formation of multicellular tumor spheroids, yielding insight into the mechanisms driving tumorigenesis.

Bioprintable 藻酸盐/明胶水凝胶 3D体外模型诱导细胞球形形成

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell ...

Core/shell Printing Scaffolds For Tissue Engineering Of Tubular Structures

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the interface with a liquid core. The latter is removed post-printing, leaving behind a hollow tube. Integrating an additive manufacturing technique (like the one described here with tailor-made [bio]inks, which structurally and biochemically mimic the native extracellular matrix [ECM]) is an important step towards advanced tissue engineering. However, precise fabrication of well-defined structures requires tailored fabrication strategies optimized for the material in use. Therefore, it is sensible to begin with a set-up that is customizable, simple-to-use, and compatible with a broad spectrum of materials and applications. This work presents an easy-to-manufacture core/shell nozzle with luer-compatibility to explore core/shell printing of woodpile structures, tested with a well-defined, alginate-based scaffold material formulation.

Presented here is a simple-to-use, core/shell, three-dimensional bioprinting set-up for one-step fabrication of hollow scaffolds, suitable for tissue engineering of vascular and other tubular structures.

管状结构组织工程的核心/壳印刷支架

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the ...

Fabrication of Engineered Vascular Flaps Using 3D Printing Technologies

Engineering implantable, functional, thick tissues requires designing a hierarchical vascular network. 3D bioprinting is a technology used to create tissues by adding layer upon layer of printable biomaterials, termed bioinks, and cells in an orderly and automatic manner, which allows for creating highly intricate structures that traditional tissue engineering techniques cannot achieve. Thus, 3D bioprinting is an appealing in vitro approach to mimic the native vasculature complex structure, ranging from millimetric vessels to microvascular networks. 
Advances in 3D bioprinting in granular hydrogels enabled the high-resolution extrusion of low-viscosity extracellular matrix-based bioinks. This work presents a combined 3D bioprinting and sacrificial mold-based 3D printing approach for fabricating engineered vascularized tissue flaps. 3D bioprinting of endothelial and support cells using recombinant collagen-methacrylate bioink within a gelatin support bath is utilized for the fabrication of a self-assembled capillary network. This printed microvasculature is assembled around a mesoscale vessel-like porous scaffold, fabricated using a sacrificial 3D printed mold, and is seeded with endothelial cells. 
This assembly induces the endothelium of the mesoscale vessel to anastomose with the surrounding capillary network, establishing a hierarchical vascular network within an engineered tissue flap. The engineered flap is then directly implanted by surgical anastomosis to a rat femoral artery using a cuff technique. The described methods can be expanded for the fabrication of various vascularized tissue flaps for use in reconstruction surgery and vascularization studies.

Engineered flaps require an incorporated functional vascular network. In this protocol, we present a method of fabricating a 3D printed tissue flap containing a hierarchical vascular network and its direct microsurgical anastomoses to rat femoral artery.

使用3D打印技术制造工程血管瓣

Engineering implantable, functional, thick tissues requires designing a hierarchical vascular network. 3D bioprinting is a technology used to create ...

<meta charset="utf8"></head><body>使用 Vessels-on-a-Plate 快速制造血管网络的 3D 生物打印

High-Throughput Bioprinting Method for Modeling Vascular Permeability in Standard Six-well Plates with Size and Pattern Flexibility

Vascular permeability is a key factor in developing therapies for disorders associated with compromised endothelium, such as endothelial dysfunction in coronary arteries and impaired function of the blood-brain barrier. Existing fabrication techniques do not adequately replicate the geometrical variation in vascular networks in the human body, which substantially influences disease progression; moreover, these techniques often involve multi-step fabrication procedures that hinder the high-throughput production necessary for pharmacological testing. This paper presents a bioprinting protocol for creating multiple vascular tissues with desired patterns and sizes directly on standard six-well plates, overcoming existing resolution and productivity challenges in bioprinting technology. A simplified fabrication approach was established to construct six hollow, perfusable channels within a hydrogel, which were subsequently lined with human umbilical vein endothelial cells to form a functional and mature endothelium. The computer-controlled nature of 3D bioprinting ensures high reproducibility and requires fewer manual fabrication steps than traditional methods. This highlights VOP's potential as an efficient high-throughput platform for modeling vascular permeability and advancing drug discovery.

<meta charset="utf8"></head><body>作者聚焦:用于高通量血管模型制造的自动化生物打印

We present a protocol for high-throughput production of vascular channels with flexible sizes and desired patterns on a standard six-well plate using 3D bioprinting technology, referred to as vessels-on-a-plate (VOP). This platform has the potential to advance the development of therapeutics for the disorders associated with compromised endothelium.

高通量生物打印方法，用于模拟具有尺寸和图案灵活性的标准六孔板中的血管通透性

Vascular permeability is a key factor in developing therapies for disorders associated with compromised endothelium, such as endothelial dysfunction in ...

将3D多细胞乳房球体直接生物打印到内皮网络

摘要

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此视频中的章节

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能体外模型

Bioprinted蜂窝构造的可行性使用三分配器笛卡尔打印机

使用3D生物印刷创建心脏组织展示球体的机械整合

微流控生物工程血管化组织和细胞器解体

微流控装置中组织模型的 Perfusable 血管网

上皮癌细胞无支架 3D 培养物中的异多细胞基质细胞驱动侵袭

Bioprintable 藻酸盐/明胶水凝胶 3D体外模型诱导细胞球形形成

管状结构组织工程的核心/壳印刷支架

使用3D打印技术制造工程血管瓣

高通量生物打印方法，用于模拟具有尺寸和图案灵活性的标准六孔板中的血管通透性

将3D多细胞乳房球体直接生物打印到内皮网络

摘要

探索更多视频

此视频中的章节

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能 体外 模型

Bioprinted蜂窝构造的可行性使用三分配器笛卡尔打印机

使用3D生物印刷创建心脏组织展示球体的机械整合

微流控生物工程血管化组织和细胞器解体

微流控装置中组织模型的 Perfusable 血管网

上皮癌细胞无支架 3D 培养物中的异多细胞基质细胞驱动侵袭

Bioprintable 藻酸盐/明胶水凝胶 3D体外模型诱导细胞球形形成

管状结构组织工程的核心/壳印刷支架

使用3D打印技术制造工程血管瓣

高通量生物打印方法，用于模拟具有尺寸和图案灵活性的标准六孔板中的血管通透性

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能体外模型