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 border="0" cellpadding="0" cellspacing="0" width="738">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Item</td>
			<td style="width:167px;"></td>
			<td style="width:169px;"></td>
			<td style="width:141px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine fibrinogen</td>
			<td>Sigma</td>
			<td>F8630-5G</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine thrombin</td>
			<td>Sigma</td>
			<td>T6634-250UN</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine aprotinin</td>
			<td>Sigma</td>
			<td>10820-25MG</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rat tail collagen I, 4 mg/mL</td>
			<td>Advanced Biomatrix</td>
			<td>5153-100MG</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodim chloride</td>
			<td>Fisher</td>
			<td>BP358-10</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS</td>
			<td>Life Technologies</td>
			<td>14190-250</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fine forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-20</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sylgard 184 silicone elastomer</td>
			<td>Corning</td>
			<td>4019862</td>
			<td>PDMS Molds</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lab tape</td>
			<td>Fisher</td>
			<td>15-901-5R</td>
			<td>PDMS Molds</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acrylic, 1/4&#34; thick</td>
			<td>McMaster-Carr</td>
			<td>8560K356</td>
			<td>PDMS Molds</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES Buffer, 1 M</td>
			<td>Sigma</td>
			<td>H3537-100ML</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI 1640 medium, powder</td>
			<td>Fisher</td>
			<td>31800-089</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcium chloride dihydrate</td>
			<td>Fisher</td>
			<td>AC423520250</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnesium chloride hexahydrate</td>
			<td>Fisher</td>
			<td>M33 500</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potassium chloride</td>
			<td>Sigma</td>
			<td>P9541-500G</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium phosphate dibasic heptahydrate</td>
			<td>Sigma</td>
			<td>S9390-500G</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glucose</td>
			<td>Sigma</td>
			<td>G5767-25G</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">OCT</td>
			<td>VWR</td>
			<td>25608-930</td>
			<td>Histology</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Frozen block molds</td>
			<td>VWR</td>
			<td>25608-916</td>
			<td>Histology</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hematoxylin</td>
			<td>Fisher</td>
			<td>3530 1</td>
			<td>Histology</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eosin Y</td>
			<td>Fisher</td>
			<td>AC152880250</td>
			<td>Histology</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fast green FCF</td>
			<td>Fisher</td>
			<td>AC410530250</td>
			<td>Histology</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Illustrator</td>
			<td>Adobe Systems</td>
			<td></td>
			<td>Vector Graphics</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Inkscape</td>
			<td>(Open Source)</td>
			<td></td>
			<td>Vector Graphics</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UCP (Universal Control Panel)</td>
			<td>Universal Laser Systems</td>
			<td></td>
			<td>Laser Cutter Interface</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PLS6.75 Laser Cutter</td>
			<td>Universal Laser Systems</td>
			<td></td>
			<td>Laser Cutter</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">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

Nanyang Technological University

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

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

磁共振弹性成像方法评价组织工程构造增长

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

科研

生物工程

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of tri-leaflet polymer valve prostheses. However, identification of a protocol for initial assessment of polymer valve hydrodynamic functionality is paramount during the early stages of the design process. Traditional in vitro pulse duplicator systems are not configured to accommodate flexible tri-leaflet materials; in addition, assessment of polymer valve functionality needs to be made in a relative context to native and prosthetic heart valves under identical test conditions so that variability in measurements from different instruments can be avoided. Accordingly, we conducted hydrodynamic assessment of i) native (n = 4, mean diameter, D = 20 mm), ii) bi-leaflet mechanical (n= 2, D = 23 mm) and iii) polymer valves (n = 5, D = 22 mm) via the use of a commercially available pulse duplicator system (ViVitro Labs Inc, Victoria, BC) that was modified to accommodate tri-leaflet valve geometries. Tri-leaflet silicone valves developed at the University of Florida comprised the polymer valve group. A mixture in the ratio of 35:65 glycerin to water was used to mimic blood physical properties. Instantaneous flow rate was measured at the interface of the left ventricle and aortic units while pressure was recorded at the ventricular and aortic positions. Bi-leaflet and native valve data from the literature was used to validate flow and pressure readings. The following hydrodynamic metrics were reported: forward flow pressure drop, aortic root mean square forward flow rate, aortic closing, leakage&#160;and regurgitant volume, transaortic closing, leakage, and total energy losses. Representative results indicated that hydrodynamic metrics from the three valve groups could be successfully obtained by incorporating a custom-built assembly into a commercially available pulse duplicator system and subsequently, objectively compared to provide insights on functional aspects of polymer valve design.

There has been renewed interest in developing polymer valves. Here, the objectives are to demonstrate the feasibility of modifying a commercial pulse duplicator to accommodate tri-leaflet geometries and to define a protocol to present polymer valve hydrodynamic data in comparison to native and prosthetic valve data collected under near-identical conditions.

协议相对液力评估的三单张聚合物阀

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

组织工程血管在小鼠模型的生成和嫁接

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

极化神经组织工程三维蚕丝胶原蛋白为基础的模式

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

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 ...

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant technique with nutritional support. Recently, there have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in early epithelization in the special environment of peristalsis. Here, we developed an artificial esophagus that can improve the regeneration of the esophageal mucosa and muscle layers through a two-layered tubular scaffold, a mesenchymal stem cell-based bioreactor system, and a bypass feeding technique with modified gastrostomy. The scaffold is made of polyurethane (PU) nanofibers in a cylindrical shape with a three-dimensional (3D) printed polycaprolactone strand wrapped around the outer wall. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. We improved the graft survival rate by applying surgical anastomosis and covering the implanted prosthesis with a thyroid gland flap, followed by temporary nonoral gastrostomy feeding. These grafts were able to recapitulate the findings of initial epithelialization and muscle regeneration around the implanted sites, as demonstrated by histological analysis. In addition, increased elastin fibers and neovascularization were observed in the periphery of the graft. Therefore, this model presents a potential new technique for circumferential esophageal reconstruction.

Esophageal reconstruction is a challenging procedure, and development of a tissue-engineered esophagus that enables regeneration of esophageal mucosa and muscle and that can be implanted as an artificial graft is necessary. Here, we present our protocol to generate an artificial esophagus, including scaffold manufacturing, bioreactor cultivation, and various surgical techniques.

用于大鼠圆周食管重建的组织工程移植

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

成纤维细胞衍生的人类工程结缔组织用于筛查应用

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

Isolation and Culture of Primary Human Gingival Epithelial Cells using Y-27632

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is an important structure of gingival tissue, especially in the repair and regeneration of periodontal tissue. Studying the functions of gingival epithelial cells has crucial scientific value, such as repairing oral defects and detecting the compatibility of biomaterials. As human gingival epithelial cells are highly differentiated keratinized cells, their lifespan is short, and they are difficult to passage. So far, there are only two ways to isolate and culture gingival epithelial cells, a direct explant method and an enzymatic method. However, the time required to obtain epithelial cells using the direct explant method is longer, and the cell survival rate of the enzymatic method is lower. Clinically, the acquisition of gingival tissue is limited, so a stable, efficient, and simple in vitro isolation and culture system is needed. We improved the traditional enzymatic method by adding Y-27632, a Rho-associated kinase (ROCK) inhibitor, which can selectively promote the growth of epithelial cells. Our modified enzymatic method simplifies the steps of the traditional enzymatic method and increases the efficiency of culturing epithelial cells, which has significant advantages over the direct explant method and the enzymatic method.

Here we present a modified method for the isolation and culture of human gingival epithelial cells by adding the Rock inhibitor, Y-27632, to the traditional method. This method is easier, less time-consuming, enhances stem cell properties, and produces larger numbers of high-potential epithelial cells both for the laboratory and for clinical applications.

使用Y-27632的原发性人类金吉瓦尔表皮细胞的隔离与培养

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is ...

Engineered Lung Tissues Prepared from Decellularized Lung Slices

There is a need for improved 3-dimensional (3D) lung models that recapitulate the architectural and cellular complexity of the native lung alveolus ex vivo. Recently developed organoid models have facilitated the expansion and study of lung epithelial progenitors in vitro, but these platforms typically rely on mouse tumor-derived matrix and/or serum, and incorporate just one or two cellular lineages. Here, we describe a protocol for generating engineered lung tissues (ELTs) based on the multi-lineage recellularization of decellularized precision-cut lung slices (PCLS). ELTs contain alveolar-like structures comprising alveolar epithelium, mesenchyme, and endothelium, within an extracellular matrix (ECM) substrate closely resembling that of native lung. To generate the tissues, rat lungs are inflated with agarose, sliced into 450 &#181;m-thick slices, cut into strips, and decellularized. The resulting acellular ECM scaffolds are then reseeded with primary endothelial cells, fibroblasts, and alveolar epithelial type 2 cells (AEC2s). AEC2s can be maintained in ELT culture for at least 7 days with a serum-free, chemically-defined growth medium. Throughout the tissue preparation and culture process, the slices are clipped into a cassette system that facilitates handling and standardized cell seeding of multiple ELTs in parallel. These ELTs represent an organotypic culture platform that should facilitate investigations of cell-cell and cell-matrix interactions within the alveolus as well as biochemical signals regulating AEC2s and their niche.

This protocol describes a method to generate reproducible, small-scale engineered lung tissues, by repopulating decellularized precision-cut lung slices with alveolar epithelial type 2 cells, fibroblasts, and endothelial cells.

由去细胞化肺切片制备的工程肺组织

There is a need for improved 3-dimensional (3D) lung models that recapitulate the architectural and cellular complexity of the native lung alveolus ex ...