作者承认来自 NIH R00 HL115123 和布朗大学工程学院的资助。他们也感谢布朗设计车间和克里斯公牛为训练和支持与激光切割机。

1. 创建矢量格式主模具设计
<ol><li>使用矢量图形程序将所需的模具几何组合成矢量格式 (请参阅材料、设备和软件表)。选择文件 |新建并创建具有 RGB 颜色格式的适当维度画布。使用左侧面板中的形状工具创建所需的几何图形: 在窗口顶部输入所需尺寸 (单击顶部的变换按钮 (如果不是最初可见), 精确定义形状大小。 注: 模具几何应允许至少6毫米的边界之间的最外层的特征和切割线, 以允许修剪后, 在模具铸造的半月板。这将使成品模具奠定冲洗与底部的文化菜肴。此外, 模具几何应考虑与将使用的激光切割机模型相关的限制, 包括切口宽度和最小特征尺寸。此处描述的工作所用的激光 (参见材料、设备和软件表) 具有0.2 毫米切缝宽度和最小蚀刻特征尺寸25µm (最大 DPI 为 1000)。模具尺寸可以选择, 以适应所需的养殖容器, 如10厘米菜, 或6或24井板。</li>
	<li>打开窗口左上角的拾色器, 并定义与激光切割软件兼容的新颜色色板。 注: 我们使用红色 (rgb 255, 0, 0) 为切割和蓝色 (rgb 0, 0, 255) 蚀刻。可以根据设计要求定义其他色板 (例如, 如果需要多个蚀刻深度)。<ol>
<li>通过选择路径, 然后为颜色选取器中的填充颜色选择路径颜色和[无]的剪切色板, 将这些色板颜色分配给裁剪路径。</li>
	</ol>
</li>
	<li>同样, 通过选择对象, 然后为颜色选取器中的路径颜色选择填充颜色和[无]的蚀刻路径, 将色板颜色分配给蚀刻路径。</li>
	<li>将设计保存为 either.ai 或. pdf 文件格式, 具体取决于矢量图形程序和激光切割器兼容性 (图 1A和辅助文件)。</li>
</ol>2. 激光切割丙烯酸主模具
<ol><li>选择负主模具材料。 注: 季度英寸厚的丙烯酸是非常适合这个应用, 因为它的兼容性与激光切割, 相对较低的成本, 和厚度, 使功能高达5.5 毫米的高度。但是, 其他满足以下要求的材料也可以使用: (i) 非反应性与二甲基硅烷固化;(二) 不含多孔/强粘接剂;(iii) 用激光切割机清洁和蚀刻;(iv) 保持玻璃的状态高达60摄氏度;(v) 与所需的最大特征高度兼容的厚度。</li>
	<li>根据制造商的规格, 准备激光切割机。确保使用足够的通风, 并将床高正确地校准到所选的负主模材料的厚度。</li>
	<li>在连接到激光切割机的计算机上的矢量图形程序中打开设计文件, 然后单击文件 |打印。确保将激光切割器设置为打印机, 并在缩放下拉菜单中选中 "不缩放", 以防止在单击打印对话框底部的 "打印" 按钮之前对设计进行失真。</li>
	<li>在激光切割机打印实用程序中, 单击右下角的设置按钮。将每英寸 (PPI) 设置的功率、速度和脉冲分配给以前选择的所有颜色, 以便为所有设计功能设置剪切/蚀刻参数。 注: 在这里, 激光切割机配备了 75 W 激光 (见材料, 设备和软件表), 使用以下设置: 100% 功率, 1% 速度, 1000 PPI 削减通过¼ "丙烯酸;100% 功率, 8% 速度和 500 PPI 蚀刻在丙烯酸的深度2.75 毫米;和100% 功率, 5% 速度, 500 PPI 蚀刻的深度3.50 毫米丙烯酸。如果激光切割器的配置或材料未知, 这些参数可以通过试用和错误的测试来确定。</li>
	<li>单击激光切割软件中的大绿色按钮, 对激光蚀刻和切割负主模具。</li>
	<li>通过去除小刷子和/或压缩空气中的残余切割碎片, 准备用于注塑的负主模具 (图 1B)。</li>
</ol>3. 为细胞或组织培养制备注塑模
<ol><li>根据生产厂家的规格, 准备一份公司的铸件组合。准备0.35 毫升/cm2的主模具;这将根据模具的特点和高度而异。</li>
	<li>在一个真空室连接到标准实验室真空线 (< 500 mmHg) 1 小时, 或者直到所有的气泡都被消除后, 将准备好的。</li>
	<li>用乙烯基或屏蔽胶带将模具的外边缘胶带 (彩色标签胶带工作良好), 使胶带延伸 > 3 毫米以上的负主模具蚀刻表面。将胶带牢固地压在模具侧面, 以防以后漏水。 注: 如果丙烯酸主模具具有通孔功能, 则可能需要将胶带应用到模具的底部 (图 1C)。</li>
	<li>在负主模的蚀刻面上倒脱气。 注: 靶材的厚度将取决于应用, 虽然厚的注塑模具底部可以使成像挑战。最小厚度为 ~ 1.5 毫米在成像考虑和容易去除模子之间的一个好平衡。</li>
	<li>将所涂覆的负主模放入真空室, 再加气为1小时, 或直至所有气泡被消除。</li>
	<li>将脱气的负主模放入60摄氏度烤箱中, 至少要6小时的固化. 确保烤箱的货架水平。另外, 允许在37°c 过夜, 或在室温下治疗72小时。</li>
	<li>如果在同一负主模具上铸造多个模具, 则使用剃刀将这些模具分开, 而它们仍在负主模具上。然后, 剥离每一个注塑模具的负主模具。确保在收集过程中, 对注塑模具进行缓慢而仔细的收集, 以防止模具功能撕裂。</li>
	<li>使用剃刀刀片, 修剪的区域与半月板从铸件, 这将防止霉菌从说谎平, 以及任何多余的材料或碎片 (图 1D)。</li>
	<li>如有必要, 准备热处理的细胞/组织铸造模具。</li>
</ol>4. 铸造胶原蛋白和纤维蛋白凝胶组织
注: 使用适当的无菌程序来维持不孕。
<ol><li>将模具粘附在所需容器的底部。将新模具粘附在非组织培养板的底部, 通过对未经处理的塑料进行牢固的压铸模 (由于其憎水性)。 注: 但是, 如果要使用组织培养处理聚碳酸酯, 或在重新使用的模具, 这往往坚持不牢固, 天然或合成胶水 (如纤维蛋白或硅胶密封胶过夜) 可用于确保附件。</li>
	<li>准备纤维蛋白原, 凝血酶, 中和的胶原蛋白铸造溶液, 使每一个期望的浓度将达到在石膏组织。把胶原蛋白放在冰上直到铸造。 注: 纤维蛋白原和凝血酶不应被允许混合, 直到紧接铸造之前。如有必要, 纤维蛋白原和凝血酶溶液也可以冷冻, 以增加聚合时间。我们使用了一个最终的纤维蛋白原浓度在0-8 毫克/毫升, 最终凝血酶浓度在 10-100 U/毫升, 和最终胶原浓度之间 0.8-2.0 毫克/毫升 (图 2)。对于工程的心脏组织, 我们经常使用心肌细胞在 12 x 106单元/毫升与1.6 毫克/毫升, 4 毫克/毫升纤维蛋白原, 20 U/毫升凝血酶 (凝血酶是立即添加在铸造之前)。优选的集中 (甚而在这些建议的范围外面) 应该经验主义地被确定。</li>
	<li>按照标准协议和并用重悬在适当浓度下收获细胞, 以达到所需的初始细胞密度。 注: 人类诱导的多潜能干细胞衍生心肌细胞的收获, 如联et 等。9将收获的细胞保存在冰上。细胞体积应在准备细胞悬浮时占去。我们使用的细胞浓度范围从 9-18 x 106细胞/mL, 虽然最佳范围预计会随细胞数量而变化 (图 2)。</li>
	<li>结合纤维蛋白原, 中和胶原蛋白, 细胞悬浮 (见步骤4.2 的例子), 以创建一个铸件组合。把铸件拌在冰上。 注: 纤维蛋白原和凝血酶的温度和浓度将决定聚合动力学;为了防止浇铸过程中的聚合, 可能需要根据将要铸造的结构的数量和体积来准备不同批次的铸件组合。</li>
	<li>对待将暴露在铸件混合的模具表面, 以减轻其疏水性 (聚硅烷憎水性将增加蛋白质吸附, 使铸造困难)。<ol>
<li>通过氧等离子体处理, 如手持高频发生器 (例如, 从利顿工程 BD-20A), 实现亲水性表面改性。将接触细胞/凝胶混合物的模具表面暴露在 3-5 s 的等离子处理中, 铸造前约5分钟。</li>
		<li>或者用表面活性剂处理, 如 Pluronic F-127 (1% 瓦特/v)10。尽可能接近铸件的表面处理, 因为极性的变化会随着时间的推移而恶化。</li>
	</ol>
</li>
	<li>在浇铸之前, 将凝血酶添加到铸件组合中, 并在不引入气泡的情况下吹打上下混合。</li>
	<li>快速工作, 将铸件混合到模具中, 用小心将混合料存入模具的所有角落和缝隙。避免在第一站以外的吸管喷射, 以防止结构内的气泡形成。</li>
	<li>重复步骤4.6 和 4.7, 必要的任何额外批次的铸件组合。</li>
	<li>将组织培养容器放置在37摄氏度孵化器中, 45 分钟。如果孵化器没有很好的加湿, 在孵化前先在霉菌周围沉积细胞介质, 以防止结构脱水。 注: 细胞培养基的类型将取决于细胞类型, 但对于工程的心脏组织结构, 我们使用 RPMI 1640 + B27 补充。</li>
	<li>45分钟后, 将结构返回到罩盖, 并在返回孵化器之前用细胞介质盖住。</li>
	<li>根据单元格和构造类型的需要, 每 48-72 小时更改单元格介质。 注意: 应根据供应商提供的建议说明进行媒体更改, 以确保最大的细胞健康。更改介质时要小心, 以免干扰构造。我们没有遇到构造浮动的问题, 但如果发生这种情况, 可以通过将高职位添加到超出媒体级别的模具设计中来防止。</li>
	<li>使用后, 用10% 漂白剂、70% 乙醇和蒸馏水依次冲洗模具。然后空气干燥, 和高压釜再使用高达10倍。</li>
</ol>5. 分析技术: 组织压实
注: 由于基质重塑而造成的压实是组织生存能力和发展的指标, 可以通过光学显微镜和图像分析方便地测量。
<ol><li>收集在模具中的结构的光学显微图像的时间增量范围从2小时到96小时后铸件。 注: 由于所需的放大倍数较低, 用摄像机安装的解剖显微镜非常适合这个应用。</li>
	<li>在图像分析套件中跟踪可见构造区域, 如 ImageJ (开源、ImageJ、nih/ij/)。</li>
	<li>分析压缩的速率和最终程度 (图 2)。</li>
</ol>6. 分析技术: 拉伸试验
注: 活动力学 (因细胞活动而产生的力或应变) 和被动力学 (在响应应用菌株或力时产生的力或应变) 是许多工程化的关键功能特性。组织, 这是特别适用于工程的心脏组织。用于分析的微机械分析仪在材料表中进行了说明。其他机械测试装置也可以类似地应用, 假设它们允许进行水合测试, 并且能够进行长度控制, 并能对范围和与组织有关的分辨率进行强制测量。对于有横截面积的组织, 按单平方毫米和刚度的顺序排列, 5 锰负荷电池是一个很好的配合。更大更硬的材料需要更大的负载单元。在测试之前, 确保对力传感器和长度控制器进行正确的校准。
<ol><li>打开长度控制器、力传感器、脉冲发生器和温度控制器。</li>
	<li>用 Tyrode 溶液填充机械测试槽 (1.8 毫米氯化钙, 1.0 毫米氯化镁, 5.4 毫米氯化钾, 140 毫米氯化钠, 0.33 毫米磷酸钠, 10 毫米 HEPES, 5 毫米葡萄糖 ddH2O, pH 值调整为 7.4)工程心脏组织。调整热电偶, 使浴缸温度达到37摄氏度。</li>
	<li>加载用于收集活动收缩数据的机械测试协议。 注: 我们已经评估了有源收缩力学使用长度步长协议, 其中5% 应变增量的长度步骤为120s 举行, 以评估应变对收缩力的影响 (图 3A)。此外, 我们还通过一个拉-断协议来评估无源机械性能, 其恒定应变率为10% 应变/分钟 (图 3B)。这两种协议都可以作为主动和被动机械分析的良好起点。</li>
	<li>在解剖范围内, 小心地将工程组织结构从其上分离出来, 使其自由浮动。 注: 经过组织压实的 Cellularized 结构很可能已经与内部模具表面分离, 在这种情况下, 需要进行最少的操作。<ol>
<li>如果压实没有导致构造释放, 使用镊子轻轻地将结构从模具的两侧和底部分开, 以避免损坏组织。</li>
	</ol>
</li>
	<li>使用镊子, 轻轻地拾起结构和转移到机械测试浴。</li>
	<li>通过解剖显微镜查看构造, 将构造的任何一端安装到附加到力传感器和拉杆臂的挂钩上。</li>
	<li>使用机器人调整拉杆臂位置, 直到构造没有施加应变。零长度控制器和力控制器。</li>
	<li>通过解剖范围对构造进行图像处理, 使构造的尺寸可以通过图像分析来测量。</li>
	<li>启动活动强制协议, 并在协议完成后保存收集的数据 (图 3)。</li>
	<li>如果需要被动的机械数据, 再调整杠杆臂, 直到有零的应用应变。在加载和启动被动力学协议 (图 3) 之前, 先将长度和强制控制器和图像再进行一次。保存收集的数据。</li>
</ol>7. 分析技术: 石蜡组织学和免疫组化
注意: 我们已经取得了最大的成功, 在成像工程组织切片使用石蜡块, 使组织形态学最好保存。该过程的所有步骤必须仔细考虑和量身定做的组织, 包括处理样品无真空或压力, 经验主义地确定适当的抗原检索方法, 并滴定的主要抗体浓度。其他技术 (如使用冻结块来准备幻灯片) 可能需要较少的时间和费用, 同时根据预期的应用程序产生足够的结果。
<ol><li>在解剖范围内, 小心地将工程组织结构从该模型中分离出来, 使用钳夹在结构之间滑动, 并将其轻轻地分开, 使其自由浮动。将这些构造转移到离心管内进行固定。 注: 在模具本身所提供的静应力条件下, 易碎构造或固定在模具中的, 可通过在培养井中改变溶液, 并在固定后将结构从模具中除去而固定在模子中。</li>
	<li>在室温下将4% 多聚甲醛 (PF) 浸没10分钟, 立即修复工程组织。估计不超过1小时每1毫米的组织厚度;不要过度修复。</li>
	<li>用磷酸盐缓冲生理盐水 (PBS) 冲洗组织。</li>
	<li>保持组织湿润, 在其上放置一滴的红色, 以染成粉红色的10三十年代, 然后用70% 乙醇冲洗。 注意: 粉红色的颜色有助于识别它在石蜡块中切片后, 将被洗走在 deparaffinization。</li>
	<li>将组织用透镜纸包裹在组织学盒中。在磁带上使用泡沫垫, 以保持组织扁平。</li>
	<li>将卡带浸入70% 乙醇, 储存在4摄氏度, 直到准备石蜡处理。</li>
	<li>在增加乙醇浓度 (2 x 30 分钟, 每 70, 95 和 100%) 的情况下, 对组织进行脱水处理。然后在三连续二甲苯浴中沐浴样品, 每30分钟。</li>
	<li>将样品浸泡在三连续的石蜡浴中, 每30分钟。</li>
	<li>预热盒式磁带, 仔细地展开并去除镜片纸上的红红染色组织, 并将石蜡渗入的组织嵌入石蜡块中。小心把样品平放在模具的底部, 以使切片更容易。</li>
	<li>根据所需的切片 (5-8 µm 厚) 和染色, 使用为工程组织优化的标准程序 (图 4) 切片组织。 注: 石蜡切片的一种替代方法是使用冰冻块, 尽管样品的形貌可能受到损害。将固定结构放置在 PBS 中的30% 蔗糖中3小时, 或直到标本平衡 (例如, 隔夜)。用 50/50 v/v 30% 蔗糖和最佳切削温度 (OCT) 冷冻介质交换溶液 1 h. 用70% 乙醇, 用干冰泥浆在塑料托盘中快速冻结, 将建筑与 OCT 冻结介质放置在冻结块中。部分块与恒温器到 10-50 µm 厚的部分。</li>
</ol>8. 分析技术: 细胞对齐
注意: 操纵组织形态和内部应力场可以调节细胞对齐, 这是许多本地组织的一个定义特征。
<ol><li>在有兴趣的几何形状的注塑模中准备工程组织结构。</li>
	<li>在养殖期结束时, 在 PBS 中洗涤构造, 固定在 4% PF (见步骤 7.2) 和收获构造, 以准备切片和组织学成像 (见7节) 或整个安装染色。 注: 整个安装染色遵循类似的步骤, 组织学/免疫组化, 但需要更长的潜伏期, 和渗透深度的染料, 抗体, 和任何成像技术 (如光穿透深度进入构造) 将需要在经验上确定的结构组成。</li>
	<li>在水平平面 (平行于模具底部的平面) 上放置组织部分, 并为感兴趣的单元格标记着色。使用 f 肌动蛋白标签, 如罗丹明, 以标记肌动蛋白应力纤维的大多数细胞类型。或者, 使用特定于单元格的标记。 注: 在工程心脏组织的情况下, 定位是由肌节染色的α actinin。</li>
	<li>手动或使用分析工具 (例如、ImageJ、MATLAB) 评估感兴趣区域中所有单元格或原子核的主轴。 注意: 对同一构造的不同区域进行分类可能很有用, 具体取决于几何图形 (如网格几何中的 "节点" 和 "捆绑" 区域), 或圆形几何图形中的 "核心" 和 "边缘"。</li>
</ol>

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作者没有什么可透露的。

定制的与组织培养相适应的注塑模几何图形在调整重要的工程组织性能方面有很大用处, 如细胞对准、扩散速率和有效刚度。此外, 这些模具对于为需要几何的分析应用程序准备组织非常有用, 如机械测试16,17。从激光切割负主模具中制备这些器件提供了一种快速、简便、低成本的利用这些工具的方法, 特别是与传统微细加工的时间和成本相比较。激光切割还允许较大的最大模具尺寸, 仅限于刀具的床尺寸。我们成功地使用了这些多功能的模具, 以执行广泛的研究与工程的心脏组织, 包括光学制图的行动潜力传播, 评估的力量生产和被动的机械性能, 和植入在大鼠心肌梗死模型中13,18。我们认识到, 除了心血管再生工程的研究生态位外, 在组织工程、药物输送、材料研究等领域的应用也很广阔。
虽然在制造模具本身方面技术上有挑战性的步骤很少, 但在创建功能组织方面也有许多关键步骤。如果在24小时后构造无法压缩周围基质, 首先通过工程组织的活性染色确定细胞的生存能力。如果细胞活力很高, 考虑改变下一组组织的结构组成。虽然结果会有很大的差异取决于细胞的数量, 我们观察到更多的压实与较高的播种密度和较低的胶原蛋白浓度。最后, 它也可能有助于补充种子细胞的细胞类型非常适合基质重塑, 如成纤维细胞, 以鼓励压实。
这些模具的一个局限性是, 它的潜力, 以吸收小疏水性分子。虽然对于我们的应用来说, 这并不成问题, 但在对这些分子的损失非常敏感的化验中可能会引起关注。在这些情况下, 可通过使用防污剂 (如 polyhydrophilic 或 polyzwitterionic 材料19) 来减轻其蛋白质吸附。另一种方法是, 可将灭菌后的可塑模成型为负母材 (从激光蚀刻正模), 用于将培养模铸造在另外一种非吸附剂材料中, 如琼脂糖。

在过去的两年中, 软光刻被广泛地用作一种制造技术来支持科学研究, 特别是在微流体、材料研究和组织工程领域, 1, 2, 3。复制成型, 其中一个对象具有一个理想的形状是由负主模创建, 提供了一个方便和低成本的方法, 生产积极的, 可用于铸造成型水凝胶。但是, 所需的负主模具通常是使用昂贵、耗时、尺寸有限的微细加工技术生产的, 需要干净的房间空间和精良的设备。虽然3D 打印提供了一种潜在的替代方案, 但由于低成本打印机的分辨率限制以及常见的3D 打印机聚合物和可抑制固化的聚硅烷之间的化学作用, 它的实用性受到了一定的限制。
激光切割和蚀刻材料, 如塑料, 木材, 玻璃和金属的切割系统最近变得大大降低成本, 因此更容易为制造研究工具。商用级激光切割机能够制造厘米刻度上的物体, 其最小特性小于25µm, 而且还需要最少的训练、专门知识和使用时间。在微流体设备的制造过程中, 对其进行了激光消融, 但据我们所知, 没有手稿描述了从激光切割负主模具中可以制造毫米和厘米级模的工艺 4.
我们使用这一技术主要是为了改善营养扩散, 细胞对齐, 和机械性能5,6,7, 以控制工程组织的形状。然而, 这种技术的通用性允许在任何领域使用模压水凝胶感兴趣, 如药物输送和材料科学研究8。利用激光切割机, 可对几乎任何几何无悬垂 (这将抑制去除没有一个多部分的模具, 这是超出本手稿的范围), 并适合在激光床的尺寸。

<table><tbody><tr><td>&lt;strong&gt;Item&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Bovine fibrinogen</td><td>Sigma</td><td>F8630-5G</td><td>Constructs</td></tr><tr><td>Bovine thrombin</td><td>Sigma</td><td>T6634-250UN</td><td>Constructs</td></tr><tr><td>Bovine aprotinin</td><td>Sigma</td><td>10820-25MG</td><td>Constructs</td></tr><tr><td>Rat tail collagen I, 4 mg/mL</td><td>Advanced Biomatrix</td><td>5153-100MG</td><td>Constructs</td></tr><tr><td>Sodim chloride</td><td>Fisher</td><td>BP358-10</td><td>Constructs</td></tr><tr><td>PBS</td><td>Life Technologies</td><td>14190-250</td><td>Constructs</td></tr><tr><td>Fine forceps</td><td>Fine Science Tools</td><td>11252-20</td><td>Constructs</td></tr><tr><td>Sylgard 184 silicone elastomer</td><td>Corning</td><td>4019862</td><td>PDMS Molds</td></tr><tr><td>Lab tape</td><td>Fisher</td><td>15-901-5R</td><td>PDMS Molds</td></tr><tr><td>Acrylic, 1/4&quot; thick</td><td>McMaster-Carr</td><td>8560K356</td><td>PDMS Molds</td></tr><tr><td>HEPES Buffer, 1 M</td><td>Sigma</td><td>H3537-100ML</td><td>Constructs</td></tr><tr><td>RPMI 1640 medium, powder</td><td>Fisher</td><td>31800-089</td><td>Constructs</td></tr><tr><td>Calcium chloride dihydrate</td><td>Fisher</td><td>AC423520250</td><td>Constructs</td></tr><tr><td>Magnesium chloride hexahydrate</td><td>Fisher</td><td>M33 500</td><td>Constructs</td></tr><tr><td>Potassium chloride</td><td>Sigma</td><td>P9541-500G</td><td>Constructs</td></tr><tr><td>Sodium phosphate dibasic heptahydrate</td><td>Sigma</td><td>S9390-500G</td><td>Constructs</td></tr><tr><td>Glucose</td><td>Sigma</td><td>G5767-25G</td><td>Constructs</td></tr><tr><td>OCT</td><td>VWR</td><td>25608-930</td><td>Histology</td></tr><tr><td>Frozen block molds</td><td>VWR</td><td>25608-916</td><td>Histology</td></tr><tr><td>Hematoxylin</td><td>Fisher</td><td>3530 1</td><td>Histology</td></tr><tr><td>Eosin Y</td><td>Fisher</td><td>AC152880250</td><td>Histology</td></tr><tr><td>Fast green FCF</td><td>Fisher</td><td>AC410530250</td><td>Histology</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Illustrator</td><td>Adobe Systems</td><td></td><td>Vector Graphics</td></tr><tr><td>Inkscape</td><td>(Open Source)</td><td></td><td>Vector Graphics</td></tr><tr><td>UCP (Universal Control Panel)</td><td>Universal Laser Systems</td><td></td><td>Laser Cutter Interface</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PLS6.75 Laser Cutter</td><td>Universal Laser Systems</td><td></td><td>Laser Cutter</td></tr><tr><td>Micromechanical Analyzer</td><td>Aurora Scientific</td><td>1530A with 5 mN load cell</td><td>Mechanical Analysis</td></tr></tbody></table>

custom engineered tissue culture molds from laser-etched masters

随着组织工程领域的不断成熟, 对各种组织参数 (包括组织形态) 的兴趣也越来越大。在千分尺上操作组织形状可以直接对细胞进行排列, 改变有效的机械性能, 并解决与养分扩散有关的限制。此外, 制备组织的容器可以对组织施加机械约束, 导致应力场, 从而进一步影响细胞和基质结构。具有高度可重现性的形状组织还具有用于体外检测的实用程序, 其中样本维度至关重要, 例如整个组织机械分析。
本手稿描述了利用激光蚀刻丙烯酸制备的负主模的替代制造方法: 这些模具与烷的性能良好, 允许设计厘米刻度和特征的尺寸尺寸小于25µm, 可快速设计和制造的低成本和最低的专业知识。最低的时间和成本要求, 使激光蚀刻模具被迅速地重复, 直到一个优化的设计是确定的, 并易于适应, 以适应任何兴趣的检测, 包括那些超出组织工程领域。

激光切割机的光学将导致蚀刻区域有非常轻微减少的尺寸, 因为蚀刻深度增加, 并导致模具墙与一个非常微妙的斜角, 由于激光束的渐狭。这将有助于去除铸造的注塑模, 但应仔细考虑, 如果非常深刻蚀刻负主模具 (> 6 毫米) 是必需的 (图 1)。
随着时间的推移, cellularized 结构将会因基体重塑而紧凑, 尽管这种情况的发生率和程度将取决于脚手架的组成、细胞负荷和培养条件。
矩阵重构可以通过周围矩阵的重组和退化 (以及新矩阵的沉积) 来实现, 但通常与随着截面面积的减小而引起的机械刚度的增加有关。只有由1.6 毫克/毫升胶原蛋白和 12 x 106心肌细胞/ml 组成的胶原蛋白结构, 我们可以看到, 通过压实试验, 在铸造 (图 19.7) 之后的2.8% 天内, 构造紧凑到四到2的初始宽度。虽然这种检测产生了3D 过程的代表性2D 逼近, 但数据收集和无损性质的简化, 使它成为研究构造开发过程的有力工具。请注意, 在细胞培养条件下, 即使在没有细胞的情况下, 胶原力学也会随着时间的推移而改变, 因为自组装和交联11。如果不存在抗纤溶, 如抑肽酶或ε-酸12, 纤维蛋白可以通过纤溶的体内和体外迅速降解。因此, 在选择构造配方时, 应考虑支架组件对组织发育的影响, 而不只是组织形成。如果最终组织尺寸对于特定的应用是重要的, 在模具设计中也必须考虑压实, 并根据细胞类型和基体组成进行经验性确定。请注意, 组织压实也可以诱发组织内的应力场, 在 cellularized 构造中可以操纵, 以鼓励细胞对齐 (图 4)。
广泛的脚手架聚合物浓度和初始细胞播种密度已被用来创建工程组织的文献, 这可以主要归因于不同的要求, 不同的细胞类型, 细胞系和应用。根据我们自己的工作与 hiPSC 心肌细胞, 我们认为, 胶原浓度1.25 毫克/毫升和播种密度为 ~ 1500万细胞/毫升是一个良好的起点13。另外, 纤维蛋白也被广泛用作心脏组织支架材料, 通常在 3-4 毫克/毫升14范围内。细胞播种密度可以根据不同的影响因素选择, 但细胞密度的本地组织提供了一个很好的参考点。还要考虑高度集中的细胞解决方案可能会对工作具有挑战性, 特别是对于小容量。对于给定的细胞群, 可以调整支架的配方;一般情况下, 当组织太脆弱或在压实时断裂时增加聚合物浓度, 当组织过于僵硬或无法压缩15时, 聚合物浓度增加。
在培养过程中, 在任何时间点进行无源机械分析之前, 可以适当的机械前提下构造样品。天然高分子水凝胶和工程组织的预适应将增加材料粘弹性试验结果的重现性, 并为该结构在临床上展示的性能提供更好的指示。应用。在开始机械评估之前, 我们在三角形波形中使用8循环10% 应变, 速度为10% 应变/分钟 (图 3)。
组织和细胞类型的特异形态学可以通过组织学和免疫组化方法来评估。然而, 我们发现, 几乎所有步骤的石蜡处理, 嵌入, 切片, 抗原检索和染色是必要的工程心脏组织相比, 电镀细胞或切片本机组织 (图4)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57239/57239fig1.jpg"> 图 1: 从激光切割丙烯酸母版中设计和制备塑料模具的过程概述.(A) 以矢量图形格式编写的模具设计。(B) 已清洗激光蚀刻丙烯酸负母。(C) 在压克力主模具的表面上铸造。(D) 导致在组织培养之前为灭菌准备的注塑模。嵌入: 顶部视图, 相同刻度。<a href="/files/ftp_upload/57239/57239fig1large.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/57239/57239fig2.jpg"> 图 2: 在区域性中构造压缩时间.(A) 在文化中, 随着时间的推移, 用三次压缩编写的矩形结构的图像。绿色覆盖表示用于计算可视化构造区域以进行图像分析的掩码。(B) 随着时间的推移, 构造区域 (构造压缩的二维度量单位) 的绘制。水平线表示平均值, 误差线表示标准偏差。对于所有组, n = 3 和 * 表示p < 0.05, 按方差分析计算。<a href="/files/ftp_upload/57239/57239fig2large.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/57239/57239fig3.jpg"> 图 3: 用于工程心脏组织的机械特性的原始痕迹.插入显示一个单一的代表性抽搐收缩跟踪 (与主剧情相同的轴)。(A) 由快速步骤所产生的主动机械响应, 后跟5% 应变增量。(B) 由拉断测试产生的被动机械响应, 其速率为10% 应变/分钟。在 Tyrode 溶液的37摄氏度浴中分析了所有样品。<a href="/files/ftp_upload/57239/57239fig3large.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/57239/57239fig4.jpg"> 图 4:各种设计的工程心脏组织结构的石蜡切片组织学图像。石蜡块组织学图像的工程心脏组织结构的各种设计染色 (A) 苏木精和伊红, (B) diaminobenzidine (抗心肌肌钙蛋白 T, 棕色) 和苏木精核 counterstain (C) picrosirius 红染色的胶原蛋白与快速绿色细胞质 counterstain, (D) 小鼠抗α actinin 抗体 (绿色) 和 4 ', 6-diamidino-2-苯基吲哚 (DAPI) 核 counterstain (蓝色)。细胞对齐方式因结构设计和组织压实而不同。在 A 中的缩放条也适用于 B 和 C。在 D 中的插入显示纹状心肌细胞。<a href="/files/ftp_upload/57239/57239fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Custom Engineered Tissue Culture Molds from Laser-etched Masters at JoVE.com

Custom Engineered Tissue Culture Molds from Laser-etched Masters

在此, 我们提出了一个快速, 简便, 低成本的方法, 以制造定制的烷模具, 可用于生产水凝胶为基础的工程组织复杂的几何。我们还描述了机械和组织学评估的结果, 对使用这种技术生产的工程心脏组织进行了研究。

由激光蚀刻大师定制的组织培养模具

As the field of tissue engineering has continued to mature, there has been increased interest in a wide range of tissue parameters, including tissue ...

custom-engineered-tissue-culture-molds-from-laser-etched-masters

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

6.4K 次观看.  Brown University. 在此, 我们提出了一个快速, 简便, 低成本的方法, 以制造定制的烷模具, 可用于生产水凝胶为基础的工程组织复杂的几何。我们还描述了机械和组织学评估的结果, 对使用这种技术生产的工程心脏组织进行了研究。

视频: Custom Engineered Tissue Culture Molds from Laser-etched Masters

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

微机械动态细胞培养平台

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

科研

生物工程

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do not have suitable autologous donor vessels due to disease progression or previous harvest. The aim of vascular tissue engineering is to develop a suitable alternative source for these bypass grafts. In addition, engineered vascular tissue may prove valuable as living vascular models to study cardiovascular diseases. Several promising approaches to engineering blood vessels have been explored, with many recent studies focusing on development and analysis of cell-based methods.2-5 Herein, we present a method to rapidly self-assemble cells into 3D tissue rings that can be used in vitro to model vascular tissues.
To do this, suspensions of smooth muscle cells are seeded into round-bottomed annular agarose wells. The non-adhesive properties of the agarose allow the cells to settle, aggregate and contract around a post at the center of the well to form a cohesive tissue ring.6,7 These rings can be cultured for several days prior to harvesting for mechanical, physiological, biochemical, or histological analysis. We have shown that these cell-derived tissue rings yield at 100-500 kPa ultimate tensile strength8 which exceeds the value reported for other tissue engineered vascular constructs cultured for similar durations (&lt;30 kPa).9,10 Our results demonstrate that robust cell-derived vascular tissue ring generation can be achieved within a short time period, and offers the opportunity for direct and quantitative assessment of the contributions of cells and cell-derived matrix (CDM) to vascular tissue structure and function.

This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly. Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days. Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.

定向细胞自我组装制造，为生物力学分析与组织工程细胞衍生的组织环

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do ...

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance1. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor2, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence2. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells3. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized1,4.
Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays5 are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment6-9.
We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further. 
Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis6. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.

A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.

癌症干细胞迁移的使用进行划分的微流控芯片和活细胞成像的评价

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

医学

Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix structure (e.g. composition, concentration, or stiffness of the matrix) vary over space, would enable a wide range of investigations concerning how these variables affect cell differentiation, migration, and other phenomena. The major challenge in creating layered matrices is maintaining the structural integrity of layer interfaces without diffusion of individual components from each layer1. Current methodologies to achieve this include photopatterning2-3, lithography4, sequential functionalization5, freeze drying6, microfluidics7, or centrifugation8, many of which require sophisticated instrumentation and technical skills. Others rely on sequential attachment of individual layers, which may lead to delamination of layers9. 
DGMP overcomes these issues by using an inert density modifier such as iodixanol to create layers of varying densities10. Since the density modifier can be mixed with any prepolymer or bioactive molecule, DGMP allows each scaffold layer to be customized. Simply varying the concentration of the density modifier prevents mixing of adjacent layers while they remain aqueous. Subsequent single step polymerization gives rise to a structurally continuous multilayered scaffold, in which each layer has distinct chemical and mechanical properties. The density modifier can be easily removed with sufficient rinsing without perturbation of the individual layers or their components. This technique is therefore well suited for creating hydrogels of various sizes, shapes, and materials.
A protocol for fabricating a 2D-polyethylene glycol (PEG) gel, in which alternating layers incorporate RGDS-350, is outlined below. We use PEG because it is biocompatible and inert. RGDS, a cell adhesion peptide11, is used to demonstrate spatial restriction of a biological cue, and the conjugation of a fluorophore (Alexa Fluor 350) enables us to visually distinguish various layers. This procedure can be adapted for other materials (e.g. collagen, hyaluronan, etc.) and can be extended to fabricate 3D gels with some modifications10.

Density Gradient Multilayered Polymerization DGMP: A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Here we describe a unique strategy for creating biocompatible, layered matrices with continuous interfaces between distinct layers for tissue engineering. Such a scaffold could provide an ideal customizable environment to modulate cell behavior by various biological, chemical or mechanical cues

密度梯度多层聚合（dGMP的）：创建多室的，可定制的一种新技术，组织工程支架

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix ...

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

芯片尺寸支架的三维细胞培养的微细

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

生物学

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

一个多室中枢神经系统的神经胶质细胞共培养的微流体平台

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

用 3 d-印刷模具制作细胞播种和组织环自组装用的自定义琼脂糖井

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

制造小鼠肌腱组合体以研究疾病中的细胞相互作用

Engineering Tendon Assembloids to Probe Cellular Crosstalk in Disease and Repair

Tendons enable locomotion by transferring muscle forces to bones. They rely on a tough tendon core comprising collagen fibers and stromal cell populations. This load-bearing core is encompassed, nourished, and repaired by a synovial-like tissue layer comprising the extrinsic tendon compartment. Despite this sophisticated design, tendon injuries are common, and clinical treatment still relies on physiotherapy and surgery. The limitations of available experimental model systems have slowed the development of novel disease-modifying treatments and relapse-preventing clinical regimes. 
In vivo human studies are limited to comparing healthy tendons to end-stage diseased or ruptured tissues sampled during repair surgery and do not allow the longitudinal study of the underlying tendon disease. In vivo animal models also present important limits regarding opaque physiological complexity, the ethical burden on the animals, and large economic costs associated with their use. Further, in vivo animal models are poorly suited to systematic probing of drugs and multicellular, multi-tissue interaction pathways. Simpler in vitro model systems have also fallen short. One major reason is a failure to adequately replicate the three-dimensional mechanical loading necessary to meaningfully study tendon cells and their function. 
The new 3D model system presented here alleviates some of these issues by exploiting murine tail tendon core explants. Importantly, these explants are easily accessible in large numbers from a single mouse, retain 3D in situ loading patterns at the cellular level, and feature an in vivo-like extracellular matrix. In this protocol, step-by-step instructions are given on how to augment tendon core explants with collagen hydrogels laden with muscle-derived endothelial cells, tendon-derived fibroblasts, and bone marrow-derived macrophages to substitute disease- and injury-activated cell populations within the extrinsic tendon compartment. It is demonstrated how the resulting tendon assembloids can be challenged mechanically or through defined microenvironmental stimuli to investigate emerging multicellular crosstalk during disease and injury.

作者聚焦：通过开发小鼠组合来了解细胞机制来推进肌腱研究

Here, we present an assembloid model system to mimic tendon cellular crosstalk between the load-bearing tendon core tissue and an extrinsic compartment containing cell populations activated by disease and injury. As an important use case, we demonstrate how the system can be deployed to probe disease-relevant activation of extrinsic endothelial cells.

由激光蚀刻大师定制的组织培养模具

摘要

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微机械动态细胞培养平台

定向细胞自我组装制造，为生物力学分析与组织工程细胞衍生的组织环

癌症干细胞迁移的使用进行划分的微流控芯片和活细胞成像的评价

密度梯度多层聚合（dGMP的）：创建多室的，可定制的一种新技术，组织工程支架

芯片尺寸支架的三维细胞培养的微细

一个多室中枢神经系统的神经胶质细胞共培养的微流体平台

用 3 d-印刷模具制作细胞播种和组织环自组装用的自定义琼脂糖井

工程肌腱组件以探测疾病和修复中的细胞串扰

工程肌腱组件以探测疾病和修复中的细胞串扰

工程肌腱组件以探测疾病和修复中的细胞串扰