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，所有生物打印应发生在37°C的无菌环境中，以最大限度地提高细胞存活率。
乳腺上皮球体可以生物印在各种生物墨水中，除了马特里格尔，包括藻酸盐和藻酸胶原蛋白混合物。我们证明球形是可行的，并保持其形态时，打印在藻酸盐为基础的生物墨水41。因此，虽然我们在这里介绍基于矩阵溶液的生物墨水的生物打印，其他更便宜和更容易使用的生物墨水也是可能的。我们还使用了其他乳腺癌细胞系，包括MCF-7和转基因MCF10A-NeuN细胞，其成功率相似，为38、41、45。替代手段也可用于创建乳房上皮球体。例如，Lee 等人使用使用 PDMS 邮票制作的水凝胶微井阵列来创建控制大小为 46的均匀大小的球形。最后，可以使用替代性内皮细胞，如肿瘤衍生的内皮细胞，而不是形成HUVEC网络，脂肪组织衍生微小瓶可以直接生物打印为3D血管结构47。
这种方法的主要局限性是控制生物打印球体位置和数量的挑战。球形必须用相对较大的喷嘴和隐身液体打印，以防止球形损伤。快速凝胶生物墨水可能更好地控制球形位置。球状物也不能计入生物墨水中，因为它们对于我们的细胞计数器来说太大了。相反，我们依赖于从在将球体从矩阵表面移开之前拍摄的相位对比图像中提取的球形计数。形成球形的替代方法可以更好地控制其数量和大小。我们每次都能以同样的方式使用细胞过滤器和培养球体，从而以中等精度控制球形数量和大小。最后的局限性是，随着时间的推移，乳房上皮细胞会从球体和内皮网络中迁移出来。替代生物墨水有可能废除这一限制。
在预形成的HUVEC网络上直接生物打印乳房上皮球体，可在短时间内创建3D体外肿瘤共生模型。然后，研究人员可以快速检查球体和血管之间的相互作用，其吞吐量更高。将来，乳房上皮球体可以生物印在注入的血管上，这将允许对流动效应的研究。此外，肿瘤衍生的器官和内皮细胞可以生物打印，通过在患者特定模型中测试药物疗效，实现精确医学。

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&#176;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&#8242;)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 &#181;m Cell Strainer</td>
			<td>Corning, Corning, NY</td>
			<td>352350</td>
			<td>Remove large or clustered spheroids</td>
		</tr>
		<tr>
			<td>CellTracker&#8482; 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&#8482; 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>Live and dead cell stain assay for cell viability</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 &#945;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>&#62;Vascular Endothelial Growth factor (VEGF165)</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乳房上皮球形可能显示细胞迁移出球体，如果在矩阵培养中维持太久。
HUVEC应在6-8小时稀疏、无血清的文化之后，自行组织成管状网络。样品将具有多细胞节点，连接由1-3个细胞并行形成。HUVEC 网络可以通过相位对比显微镜或共分显微镜进行成像，如果它们标有细胞跟踪器和 Hoescht（图 2）。ImageJ血管生成分析仪可用于量化网络路口、段和分支。如果留在无血清介质中超过16小时，HUVEC网络将死亡。
当乳房上皮球体被生物印在HUVEC网络上时，球体和网络都应该保持其原始形态至少24小时。MCF10A乳房上皮球体将作为圆形物体出现，直接粘附于内皮网络，而MDA-MB-231乳房上皮球体将显得更无定形，但仍连接或接近内皮网络（图3）。当与乳房上皮球体共同培养时，将维持HUVEC网络。对于超过24小时的共生细胞，乳房上皮细胞可能会从球体和内皮网络迁移。根据我们的经验，这发生在肿瘤，而不是非肿瘤性乳房上皮细胞38更早。我们以前曾证明使用生物打印的共生体，药物测试可以早在球形生物打印后2小时启动，例如测试球形粘附到内皮网络45。我们还表明，3D乳房球形对像Paclitaxel这样的抗癌药物的耐药性比作为单个细胞或共培养41，45打印时更强。在没有生物打印球体的情况下，HUVEC网络控制井本身无法保持其网络形态，并在16小时后死亡。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61791/61791fig01.jpg"> 图1:乳腺上皮球体的代表性胸腔显微镜图像。MCF10A球形被标记为集成+6（绿色）和核（蓝色）。当球体以空心中心（内嵌）出现圆形并在外缘极化时，细胞表型可以在生物打印后得到确认。MDA-MB-231球形被标记为作用素（绿色）和核（蓝色）。当球体在没有空心中心的情况下不规则地形成，并且细胞过程侵入周围的矩阵时，细胞表型可以被证实。缩放条 = 50μm。 <a href="https://www.jove.com/files/ftp_upload/61791/61791fig01large.jpg" target="_blank">请单击此处查看此数字的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61791/61791fig02.jpg"> 图2:按相对比和共分显微镜分组的HUVEC网络代表性图像。HUVEC 网络显示为小型多细胞节点，带有连接节点的细胞线。对于共分显微镜，细胞被标记为细胞跟踪器红色和霍施特核（蓝色）。缩放条 = 100μm。 <a href="https://www.jove.com/files/ftp_upload/61791/61791fig02large.jpg" target="_blank">请单击此处查看此数字的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61791/61791fig03.jpg"> 图3:与HUVEC网络共同培养的乳腺上皮球体的代表性图像。MCF10A球形，标记为整形+6（绿色）和核（蓝色），保持圆形，并显示直接粘附于内皮网络。MDA-MB-231球形，标记为作用素（绿色）和核（蓝色），看起来无定形，但仍在内皮网络附近或上。共同培养在生物打印后至少24小时保持这种形态，之后乳房细胞可能会沿着内皮管迁移。缩放条 = 50μm。 <a href="https://www.jove.com/files/ftp_upload/61791/61791fig03large.jpg" target="_blank">请单击此处查看此数字的较大版本。</a>

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

Nanyang Technological University

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

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

一个简单的悬滴细胞生成三维球体文化协议

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

科研

生物工程

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

组织工程:建设多细胞三维支架的层状细胞片交货

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

Modeling Ovarian Cancer Multicellular Spheroid Behavior in a Dynamic 3D Peritoneal Microdevice

Ovarian cancer is characterized by extensive peritoneal metastasis, with tumor spheres commonly found in the malignant ascites. This is associated with poor clinical outcomes and currently lacks effective treatment. Both the three-dimensional (3D) environment and the dynamic mechanical forces are very important factors in this metastatic cascade. However, traditional cell cultures fail to recapitulate this natural tumor microenvironment. Thus, in vivo-like models that can emulate the intraperitoneal environment are of obvious importance. In this study, a new microfluidic platform of the peritoneum was set up to mimic the situation of ovarian cancer spheroids in the peritoneal cavity during metastasis. Ovarian cancer spheroids generated under a non-adherent condition were cultured in microfluidic channels coated with peritoneal mesothelial cells subjected to physiologically relevant shear stress. In summary, this dynamic 3D ovarian cancer-mesothelium microfluidic platform can provide new knowledge on basic cancer biology and serve as a platform for potential drug screening and development.

To study ovarian tumor progression in a physiologically relevant model, multicellular spheroids were cultured in a microdevice under simulated fluid flow. This dynamic 3D model emulates the intraperitoneal environment with the cellular and mechanical components where ovarian cancer metastasis occurs.

模拟卵巢癌多细胞球体行为动态三维微型腹膜

Ovarian cancer is characterized by extensive peritoneal metastasis, with tumor spheres commonly found in the malignant ascites. This is associated with ...

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 ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioink Precellularization 无源混合装置打印软骨及皮肤组织类似物的应用

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Lab-on-a-CD Platform for Generating Multicellular Three-dimensional Spheroids

A three-dimensional spheroid cell culture can obtain more useful results in cell experiments because it can better simulate cell microenvironments of the living body than two-dimensional cell culture. In this study, we fabricated an electrical motor-driven lab-on-a-CD (compact disc) platform, called a centrifugal microfluidic-based spheroid (CMS) culture system, to create three-dimensional (3D) cell spheroids implementing high centrifugal force. This device can vary rotation speeds to generate gravity conditions from 1 x g to 521 x g. The CMS system is 6 cm in diameter, has one hundred 400 &#956;m microwells, and is made by molding with polydimethylsiloxane in a polycarbonate mold premade by a computer numerical control machine. A barrier wall at the channel entrance of the CMS system uses centrifugal force to spread cells evenly inside the chip. At the end of the channel, there is a slide region that allows the cells to enter the microwells. As a demonstration, spheroids were generated by monoculture and coculture of human adipose-derived stem cells and human lung fibroblasts under high gravity conditions using the system. The CMS system used a simple operation scheme to produce coculture spheroids of various structures of concentric, Janus, and sandwich. The CMS system will be useful in cell biology and tissue engineering studies that require spheroids and organoid culture of single or multiple cell types.

We present a motor-powered centrifugal microfluidic device that can cultivate cell spheroids. Using this device, spheroids of single or multiple cell types could be easily cocultured under high gravity conditions.

用于生成多细胞三维球体的 CD 实验室平台

A three-dimensional spheroid cell culture can obtain more useful results in cell experiments because it can better simulate cell microenvironments of the ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

明胶美沙克里洛醇水凝胶生物油墨3D生物印刷方案

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...

3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also respond to neural injuries and act to protect the tissue from degenerative events. In vitro studies of astrocytes&#39; functionality are important to elucidate the mechanisms involved in such events and contribute to developing therapies to treat neurological disorders. This protocol describes a method to biofabricate a neural-like tissue structure rich in astrocytes by 3D bioprinting astrocytes-laden bioink. An extrusion-based 3D bioprinter was used in this work, and astrocytes were extracted from C57Bl/6 mice pups&#39; brain cortices. The bioink was prepared by mixing cortical astrocytes from up to passage 3 to a biomaterial solution composed of gelatin, gelatin-methacryloyl (GelMA), and fibrinogen, supplemented with laminin, which presented optimal bioprinting conditions. The 3D bioprinting conditions minimized cell stress, contributing to the high viability of the astrocytes during the process, in which 74.08% &#177; 1.33% of cells were viable right after bioprinting. After 1 week of incubation, the viability of astrocytes significantly increased to 83.54% &#177; 3.00%, indicating that the 3D construct represents a suitable microenvironment for cell growth. The biomaterial composition allowed cell attachment and stimulated astrocytic behavior, with cells expressing the specific astrocytes marker glial fibrillary acidic protein (GFAP) and possessing typical astrocytic morphology. This&#160;reproducible protocol provides a valuable method to biofabricate 3D neural-like tissue rich in astrocytes that resembles cells&#39; native microenvironment, useful to researchers that aim to understand astrocytes&#39; functionality and their relation to the mechanisms involved in neurological diseases.

Here we report a method of 3D bioprinting murine cortical astrocytes for biofabricating neural-like tissues to study the functionality of astrocytes in the central nervous system and the mechanisms involving glial cells in neurological diseases and treatments.

用于工程神经样组织的小鼠皮质星形胶质细胞的3D生物打印

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also ...

Affordable Oxygen Microscopy-Assisted Biofabrication of Multicellular Spheroids

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue assembling units for biofabrication. While the main power of the spheroid model is in mimicking physical-chemical gradients at the tissue microscale, the real physiological environment (including dynamics of metabolic activity, oxygenation, cell death, and proliferation) inside the spheroids is generally ignored. At the same time, the effects of the growth medium composition and the formation method on the resulting spheroid phenotype are well documented. Thus, characterization and standardization of spheroid phenotype are required to ensure the reproducibility and transparency of the research results. The analysis of average spheroid oxygenation and the value of O2 gradients in three dimensions (3D) can be a simple and universal way for spheroid phenotype characterization, pointing at their metabolic activity, overall viability, and potential to recapitulate in vivo tissue microenvironment. The visualization of 3D oxygenation can be easily combined with multiparametric analysis of additional physiological parameters (such as cell death, proliferation, and cell composition) and applied for continuous oxygenation monitoring and/or end-point measurements. The loading of the O2 probe is performed during the stage of spheroid formation and is compatible with various protocols of spheroid generation. The protocol includes a high-throughput method of spheroid generation with introduced red and near-infrared emitting ratiometric fluorescent O2 nanosensors and the description of multi-parameter assessment of spheroid oxygenation and cell death before and after bioprinting. The experimental examples show comparative O2 gradients analysis in homo- and hetero-cellular spheroids as well as spheroid-based bioprinted constructs. The protocol is compatible with a conventional fluorescence microscope having multiple fluorescence filters and a light-emitting diode as a light source.

The protocol describes high-throughput spheroid generation for bioprinting using the multi-parametric analysis of their oxygenation and cell death on a standard fluorescence microscope. This approach can be applied to control the spheroids viability and perform standardization, which is important in modeling 3D tissue, tumor microenvironment, and successful (micro)tissue biofabrication.

经济实惠的氧气显微镜辅助多细胞球体生物制造

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue ...

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.