我们感激地感谢埃里卡 Stults 博士 (学术研究 & 应用科学家, 信息技术服务) 为她的帮助与3D 印刷, 阿曼达 Zoë Reidinger, 博士, 克里斯 Nycz, 和凯伦, 法医, 为他们对模具设计的投入,凯西 Suqui 和珍妮弗. 曼为他们的协助测试模具设计, 和迈克尔 O ' 柯弗 ' 为他的协助拍摄。 这项工作得到了 NSF IGERT DGE 1144804 (水利部)、nih R15 HL097332 (水利部、TAH)、NSF 拉吴 EEC0754996 (BA)、nih 1R01 EB023907 (水利部) 和 nih R15 HL137197 (水利部) 的支持。
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1. 准备3维印刷模具
<ol><li>在所需尺寸内准备模具的 CAD 绘图。</li>
	<li>将 CAD 文件发送到高分辨率3D 打印机。选择合适的塑料印刷材料, 有光泽的完成。</li>
	<li>印刷后, 用洗涤剂和水彻底清洗模具。</li>
</ol>2. 压铸底片
<ol><li>根据平衡量来测量所需的。对于具有60毫米直径和2毫米井柱的模具, 请使用25克。</li>
	<li>在 1:10 (w/w) 比的基础上添加硫化剂。</li>
	<li>用力搅拌, 直到两个组分完全结合。不充分的混合可能导致不完全固化的聚硅烷, 导致一个 "俗气" 的表面。</li>
	<li>将一张实验室胶带放在3维印刷模具的边界周围, 以便在印刷井上方形成一层基片。</li>
	<li>将该混合料倒入模具中, 将模具放入真空室, 直至释放所有气泡。</li>
	<li>治疗 2–4 h 50 摄氏度, 或直到凝固足够的去除。</li>
	<li>取出实验室胶带, 并仔细提取该底片。</li>
	<li>在60摄氏度的时候, 将其孵化为额外的1小时 (从 3 d-印刷模具中取出) 以确保模具完全固化。</li>
	<li>用洗涤剂和水彻底清洗, 去除残留物。不充分的洗涤可能导致不良的圆环形成为第一个用途的反。</li>
</ol>3. 制备琼脂糖井
<ol><li>在播种细胞前1天准备琼脂糖水井。</li>
	<li>在 DMEM 和高压釜中制作 2% (w/v) 琼脂糖溶液。</li>
	<li>吸管4毫升熔融琼脂糖成一个蒸压的甲基硅烷阴性。然后吸管琼脂糖直接进入了其底片的帖子。去除任何气泡与吸管尖端, 因为气泡可能会导致不均匀性在井维度。</li>
	<li>注: 请勿溢出模具;琼脂糖的顶端表面必须是平的, 因此琼脂糖井将是水平的, 当去除从聚硅烷和安置入6井培养板材。剩余的琼脂糖可以重新蒸压, 并再次使用, 虽然不超过一次。</li>
	<li>在琼脂糖冷却以后 (大约10分钟为4毫升琼脂糖; 更大的模子可能需要更长的冷却时间), 小心地分离琼脂糖井从用钝钳和转移入一个6井板材的井。</li>
	<li>在完全培养基中浸泡琼脂糖井, 并在使用前在37摄氏度的孵化器中平衡过夜。</li>
</ol>4. 种子组织环
<ol><li>培养大鼠主动脉平滑肌细胞21 (RaSMCs) 在 DMEM 含有10% 胎牛血清 (血清), 1% l-谷氨酰胺, 1% 非必需氨基酸, 1% 丙酮酸钠, 1% 青霉素-链霉素, 直到它们达到70% 汇合。 注意: 细胞应该培养在 5% CO2和37°c 在 15 cm 组织培养板材。</li>
	<li>模具平衡后 (3 节), 准备 RaSMCs 播种。</li>
	<li>用5毫升的磷酸盐缓冲生理盐水 (PBS) 冲洗两次盘子。</li>
	<li>添加3毫升0.25% 胰蛋白酶每15厘米培养皿。把盘子移到一个37摄氏度的 2–3, 或者直到细胞从盘子里抬起。</li>
	<li>中和胰蛋白酶的同等体积 (每板3毫升) 完全培养基。并用重悬细胞彻底分解成团簇。</li>
	<li>用台盼蓝染料稀释细胞悬浮1:1 的整除, 用 hemocytometer 计数细胞。</li>
	<li>离心机细胞悬浮液总体积为5分钟, 在 200 x g 到颗粒细胞。</li>
	<li>每毫升1000万细胞浓度的并用重悬细胞;这将导致每50µL 50万个细胞。 注意: 不同的细胞类型可能需要不同的细胞浓度。</li>
	<li>从琼脂糖霉菌中吸取所有培养基。小心把所有介质从单个井中取出, 但不要刺穿水井的底部。</li>
	<li>吸管50µL 悬浮入每个井。</li>
	<li>小心添加2毫升新鲜培养基周围的琼脂糖霉菌。小心不要让中溢入琼脂糖的水井。在孵化室过夜 (大约16小时)。</li>
	<li>夜间孵化后, 从模具外部吸入介质, 并在6井板的每个井中加入4.5 毫升的新鲜培养基, 使模具和环完全浸没。每日更改媒体。</li>
</ol>

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作者没有什么可透露的。

在这里, 我们提出了一个通用的方法, 快速制造自组装的组织环, 易于定制的尺寸使用3D 印刷。我们的方法类似于在 Svoronos et al中报告的。6,其中3个 d-印制的蜂窝状和狗骨形蜡模被用来铸造的。然而, 模具已被修改, 以包含几个独特的设计功能。凹槽 (图 4A(1)) 提供了模具的方向, 允许每个圆环分别进行标记和监视。中心孔 (图 4A(2)) 有助于改善介质向油井的扩散。CAD 文件编号直接打印在模具上;因此, 在每个标签上都标有版本号和 post 直径 (图 4A3)。锥形外墙 (图 3(1), 5 °), 在井槽的顶部 (图 3(2), 45 °) 和中央孔 (图 3(3), 5 °), 使从 3 d 打印模具中移除该底片更容易, 琼脂糖井更容易从该底片 (图 4A(2), A (4)) 中删除。
我们通过制作各种直径和细胞类型的自组装环形组织, 包括主要人体平滑肌细胞 (SMCs)18,22, 大鼠主动脉 SMCs7 , 展示了该系统的通用性。,23, 人骨髓间充质干细胞 (hMSCs)13, SMCs 来源于诱导多能干细胞 (iPSCs)11 (表 1)。在目前的工作中, 我们正在评估从其他细胞类型, 如内皮细胞的形成环, 并融合不同大小的软骨环, 用于气管置换的潜在应用。除了完全的细胞衍生结构, 我们也使用这个系统制造环与合并交联明胶微球13,22。在自组装过程中, 微球可以在组织环内加入, 以提供额外的机械强度, 或者用于生长因子的局部传递13,22。
在制作组织环时, 可能需要对不同细胞类型进行细胞数的优化。根据单元格的大小和类型, 最小单元格数可能会有所不同。例如, 从 iPSCs 派生的 hSMCs 为60万个单元格/环11, hMSCs 和主 hSMCs 在40万个单元格/环13、22上播种, 鼠主动脉 SMCs 在50万个单元格/环18上播种。槽尺寸也可能影响圆环形成和圆环形成所需的最小单元数24。对于人类细胞和 3 d-印刷模具的研究, 使用了槽宽2毫米。原来的聚碳酸酯模具的槽宽为3.75 毫米, 需要 75万 hSMCs 形成2毫米细胞环18。随着槽宽的降低, 我们能够将环状形成所需的细胞数量减少 46%, 每环的40万个细胞为25。在表 1中汇总了每环所播种的单元格数量。
在选择3维印刷材料时, 需要考虑许多因素。由于其通常固化在60摄氏度, 3 d-印刷材料必须有足够高的熔化温度, 以避免损害在进行了在进行中。本研究中使用的材料的熔化温度 (一种专有材料, 请参阅材料表) 不可用。然而, 当烘烤60°c 为1小时, 我们观察到的材料开始产生一种气味。因此, 我们决定降低固化温度为50摄氏度, 并增加固化时间, 以烘烤的, 而不损害 3 d-印刷材料。如果模具被修改以形成更大的, 则固化时间的调整可能是必要的。另一种固化期在60摄氏度后, 从 3 d-印刷模具去除, 防止最终的反式树脂的残余俗气, 同时限制了 3 d-印刷模具的温度暴露。请注意, 有些材料抑制了对甲基硅烷的固化, 因此确保所选材料与其兼容。最后, 还必须考虑模具材料的毒性。虽然 3 d 打印模具将不会与细胞直接接触, 有可能在固化过程中, 从模具中的一些残留物可能被转移到基硅烷阴性。我们发现, 非常彻底的洗涤与洗涤剂是足以消除任何残留从该公司的负面。然而, 我们以前观察到, 不充分的洗涤导致了在琼脂糖井的圆环形成不良为最初的少量用途的对此。使用来自其他3维印刷材料的滤液可能需要额外的调查, 以验证洗涤剂是否足以去除霉菌残留物, 包括任何潜在的。定期测试也可能是必要的, 因为它可能的重复加热循环 (即使到50°c) 可能会损坏模具随着时间的推移, 并导致在重复使用后残留增加。到目前为止, 我们已经使用了一个 3 d 打印模具产生超过30个, 已被用于成功地产生组织环的一个。
总体而言, 3D 打印允许更多功能的制造琼脂糖模具比加工的聚碳酸酯。它提供了更高的分辨率比可能与模具, 模具设计不受限制的工具的尺寸可用。这允许更大的自定义, 并增加功能, 如削尖, 可能是不可能的加工。除了圆环6、17之外, 该系统还可用于在其他形状中制作自组装组织。利用环形制作方法, 我们开发了各种细胞类型和大小的组织环, 用于气管组织工程中潜在的应用13, 工程血管7, 血管疾病的建模11。

细胞自组装方法制造组织工程化血管是一种替代脚手架的方法。自组装, 无支架组织可能有更大的细胞密度, 增强基质沉积和强度, 并改善生物学功能相比, 基于脚手架的组织1,2,3,4.然而, 在没有使用外源脚手架支持的情况下, 形成3D 组织是一个挑战。有些方法将单元格层的图层融合在一起, 形成较厚的构造, 尽管此过程可能耗费大量时间, 而且劳动密集型5。或者, 可以将单元格播种到非粘附模具中, 并允许聚合成球体、圆环和其他组织形状6,7,8。
自组装组织环要求较少的细胞, 较短的培养时间和较少的试剂比较大的管状工程组织, 但仍可进行机械测试, 组织学检查, 或用于收缩力和其他功能测试7,9,10,11. 因为它们可以快速制作和易于测试, 组织环是筛选大量的文化参数的理想选择, 并有潜力作为疾病模型11或药物筛选的工具12。此外, 环可以融合成更复杂的组织结构, 如血管或气管7,13, 和环可能更完全融合比其他形状, 如球体14,15。
琼脂糖因其生物相容性、渗透性和非细胞粘接性能而被广泛用作制造自组装组织的模具材料。例如, Norotte et从挤出棒中制造了琼脂糖模具, 这使得对模具形状和所需的专用设备的有限控制达到了15。棕褐色et将海藻酸盐滴作为建筑单位, 制造出各种形状 (金字塔, 方形)16的定制水凝胶模具。然而, 海藻酸盐球体 (300 µm) 的大直径导致了低分辨率的特征。这种低分辨率可能导致不均匀的模具表面, 从而对细胞聚集一致性产生不利影响。或者, 琼脂糖可以被投进聚合物底片, 以创建具有平滑特性和特定尺寸的非粘附模具6,7,17。
我们以前报告了一个系统, 制造自定义环状琼脂糖细胞播种井从一个在碾磨聚碳酸酯模具7,18。琼脂糖被倒进了一个阴性, 允许设置7,18。然后将细胞播种到琼脂糖阱中, 它们聚集在不到24小时的7,18中, 形成自组装的无支架组织环。耐高压, 可多次重复使用, 柔软灵活, 易于去除固化琼脂糖。最初在 Gwyther et中报告此系统时。7中, 从碾磨聚碳酸酯模具 (图 1A) 中浇铸出了其底片。琼脂糖浇铸后, 细胞播种井分别被切出并放入12井板7,18的水井中。最近对设计进行了修改, 使单琼脂糖模产生5环, 适合于6井板的井中, 无需切断单个油井, 减少生产每枚戒指所需的中和琼脂的数量 (图 1B)。使用较小的细胞播种槽宽度, 以减少所需的种子细胞数量, 以实现环形成。尽管有这些变化, 模具的分辨率和定制也被限制在可用的标准立铣刀尺寸上, 而且 micromilling 的成本也很高。此外, 计算机数控 (CNC) 加工可以耗费时间和繁琐, 因为需要在大量使用的定制设备上预留时间, 增加计算机辅助制造 (CAM) 软件来转换计算机辅助设计 (CAD) 文件到可编程刀具路径, 以及在加工过程中聚碳酸酯零件的可靠夹具。
在本研究中, 我们考察了3D 印刷作为数控加工的替代品的使用。3D 印刷广泛用于工程定制植入物, 制造脚手架材料, 并用于直接打印细胞和组织球体15,19,20。我们使用了高分辨率的3D 打印机, 和专门的3D 打印材料, 使我们能够打印一个刚性模具, 光滑, 光泽表面完成 (见材料表)。我们的技术允许制造高可定制, 高分辨率的塑料模具, 可以用于铸造中的底片和琼脂糖井。在图 1中总结了设计迭代。模具设计进一步修改, 在 3 d-印刷模具版本, 包括锥形外墙和中心孔, 以减轻从 3 d-印刷模具和琼脂糖的反胶油的底片去除。这些锥形特征不能用标准加工工艺实现。在这个迭代中, 从井底到模具底部的距离增加了, 从而在柱下形成了一个较厚的琼脂糖基, 以减少在琼脂糖清除过程中出现断柱的风险。模具和环形制造过程在图 2中以示意图的方式显示。

<table><tbody><tr><td>SeaKem LE Agarose</td><td>Lonza</td><td>50040</td><td></td></tr><tr><td>PDMS</td><td>Dow Corning</td><td>Sylgard 184</td><td></td></tr><tr><td>DMEM</td><td>Corning Cellgro</td><td>15-017-CV</td><td></td></tr><tr><td>VeroWhite</td><td>StrataSys</td><td>RGD835</td><td></td></tr><tr><td>3D printer</td><td>StrataSys</td><td>Objet 260 Connex</td><td></td></tr><tr><td>DMEM</td><td>Corning Cellgro</td><td>15-017-CV</td><td></td></tr><tr><td>FBS</td><td>Thermo Fisher</td><td>16000069</td><td></td></tr><tr><td>L-glutamine</td><td>Corning Cellgro</td><td>25-015-CI</td><td></td></tr><tr><td>Non-essential amino acids</td><td>Corning Cellgro</td><td>25-025-CI</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Corning Cellgro</td><td>25-000-CI</td><td></td></tr><tr><td>Pen-strep</td><td>Corning Cellgro</td><td>30-002-CI</td><td></td></tr><tr><td>Trypsin</td><td>Corning Cellgro</td><td>25-053-CI</td><td></td></tr><tr><td>Trypan blue</td><td>Corning Cellgro</td><td>25-900-CI</td><td></td></tr><tr><td>PBS</td><td>Lonza</td><td>17-516F</td><td></td></tr><tr><td>6-well plate</td><td>Corning</td><td>353046</td><td></td></tr><tr><td>WKY 3M-22 rat aortic smooth muscle cells</td><td>Provided by T. Wight [ref 21]</td><td>N/A</td><td></td></tr></tbody></table>

fabrication of custom agarose wells for cell seeding and tissue ring self-assembly using 3d-printed molds

工程组织正在临床上用于组织修复和置换, 并正在开发作为药物筛选和人类疾病模型的工具。自组装组织提供优于脚手架的组织工程, 如增强基质沉积, 强度和功能。然而, 没有在支架内或内的种子细胞制造3D 组织的可用方法很少。以前, 我们开发了一个系统, 以制造自组装组织环的种子细胞, 以非粘性琼脂糖水井。在加工聚碳酸酯模具中首先铸造了烷的阴性, 然后将琼脂糖凝胶在其阴性形成环状细胞播种井。然而, 这种方法的通用性受限于用于加工聚碳酸酯模具的工具的解决。在这里, 我们证明, 3 d-印刷塑料可以作为替代加工聚碳酸酯的制造新的底片。3 d-印刷模具和修订模具设计是更简单的使用, 廉价的生产, 并要求大大减少琼脂糖和每细胞播种良好。我们已经证明, 由此产生的琼脂糖水井可以用来创建自组装的组织环与定制的直径从各种不同的细胞类型。环可以用于机械, 功能, 和组织学分析, 或制造更大和更复杂的管状组织。

该系统允许简单, 定制的琼脂糖细胞播种井的制作, 使细胞自组装的环形3D 工程组织。3D. 在模具设计中, 比起加工聚碳酸酯, 尺寸受可用刀具尺寸的限制, 使其分辨率和灵活性更高。有了高分辨率的3D 打印机, 可以打印0.254 毫米厚的墙壁, 槽尺寸仅受打印机分辨率的限制 (这些研究的15.2 µm 分辨率)。通常, 不到0.3 毫米的 CNC 端铣刀在商业上是不可用的, 因此不可能创建较小宽度的槽。微铣可以产生较小的功能, 虽然所需的设备可能是昂贵或难以进入许多生物医学研究实验室。槽尺寸和曲率也受立铣刀尖端形状的限制。此外, 有角或锥形墙的功能是不可能的, 小的功能可能会打破在加工过程中。
CAD 图纸可以很容易地修改, 以生产 3 d 印刷模具和可定制尺寸的负面因素。图 3描述了与以前的设计迭代相比, 当前 2 mm 后 3 d 打印模具的尺寸。过程是低廉的;2 mm, 5 环 3 d 打印模具成本44.67 美元至3D 打印, 每部分可用于创建多个的反式底片。到目前为止, 我们已经创造了超过30个从一个单一的 3 d 打印模具底片。每一个负数要求25克的0.11 美元每克 (每一个2.75 美元的%)。根据使用频率的不同, 可以用洗涤剂、蒸压和再用数年的方法清洗每一个。与原始设计18相比, 紧凑型 3 d 打印模具的使用率要低得多, 每 2 mm 组织环 (图 1E) 中的琼脂糖。
平衡琼脂糖阱中的细胞聚集在不到24小时内形成组织环, 其大小取决于琼脂糖阱的尺寸。在这里, 我们证明了自组装大鼠平滑肌细胞环可以在井中制造 2, 4, 或12毫米直径员额 (图 4)。虽然这项研究中的环只培养了3天, 我们以前培养的 hSMC 环长达2周22, hMSC 软骨环长达3周13。如前所述, 环可以作为人体组织的 3D体外模型来进行组织功能的定量评估11和机械强度13,22, 也可以作为模块化的建筑单位, 以生成管状组织构造7,13。从这些细胞类型设计的组织环的细胞播种条件和功能属性在表 1中进行了总结。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56618/56618fig1.jpg"> 图 1: 聚碳酸酯模具设计.最初, 15 井模具被加工成聚碳酸酯 (A)。后来的版本有一个更紧凑的设计 (B), 5 口井设计, 以适应单一的聚碳酸酯模具, 并在一个井的6井板。在这里, 我们修改了这个设计, 以使用 3 d 印刷塑料作为一个更可定制的替代加工聚碳酸酯 (C)。在 (D) 中显示的琼脂糖井是从最初设计的18 (左) 与当前紧凑型设计 (右) 相比的, 它需要大大减少琼脂糖, 不需要人工分离水井。每种设计迭代所需的聚硅烷和琼脂糖数量 (E) 中显示。中心柱直径为2毫米。模子在 (A) 是 6 cm x 12 cm, (B) 有 5 cm 直径, 并且 (C) 有 6 cm 直径。缩放条 (D) = 3 厘米.<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56618/56618fig1large.jpg" target="_blank">请单击此处查看此图的更大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56618/56618fig2.jpg"> 图 2: 自组装组织环的制作.一个 3 d 打印模具被用来铸造一个负极, 然后用于铸造琼脂糖水井 (a)。然后将细胞直接播种到琼脂糖阱中, 它们聚集在不到24小时内形成组织环 (B)。(B) 中的虚线显示了井轮廓。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56618/56618fig2large.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/56618/56618fig3.jpg"> 图 3: 3 维打印模具 CAD 绘图的横断面视图.显示槽宽度 (A)、槽高度 (B)、中心孔 (C)、总直径 (D)、外唇 (E) 和外墙高度 (F) 的尺寸。外墙 (1)、水井 (2) 的上部部分和中心孔 (3) 呈锥形 (在5°角为1和 3, 45 °为 2), 以提高对其负移除的方便性。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56618/56618fig3large.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/56618/56618fig4.jpg"> 图 4: 带有相应的自组装 SMC 和 2 (A)、4 (B) 和 12 (C) 毫米柱径的 CAD 绘图.在 (A) 中, (1) 指示提供方向的凹槽 (2) 表示用于改善灌注的中心孔 (3) 是一个文件号, 它直接印在了该外墙的底片上, (4) 显示了外层壁, 这是锥形的, 以缓解其负移除。琼脂糖井中的刻度条 = 1 厘米.<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56618/56618fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>单元格类型</td>
			<td>每圈种子细胞</td>
			<td>环形直径</td>
			<td>文化持续时间</td>
			<td>执行的功能分析</td>
		</tr>
<tr>
<td>大鼠 SMC</td>
			<td>0.5、1或 3 x 106
</td>
			<td>2、4或12毫米</td>
			<td>3天</td>
			<td>当前研究;N/A</td>
		</tr>
<tr>
<td>
iPSC 派生的 SMC11
</td>
			<td>0.6 x 106
</td>
			<td>2毫米</td>
			<td>17天</td>
			<td>收缩力试验、单轴拉伸试验、组织学分析</td>
		</tr>
<tr>
<td>
人工 SMC22
</td>
			<td>0.4 x 106
</td>
			<td>2毫米</td>
			<td>2、7或14天</td>
			<td>单轴拉伸试验、组织学分析</td>
		</tr>
<tr>
<td>
人类 MSC14
</td>
			<td>0.4 x 106
</td>
			<td>2毫米</td>
			<td>21天</td>
			<td>单轴拉伸试验、组织学分析、环形融合和管压缩试验, 分化为软骨组织</td>
		</tr>
</tbody></table>表 1: 不同细胞类型的环形加工所需的细胞数。

Watch this Scientific Journal Video about Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds at JoVE.com

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

该协议描述了一个平台, 用于制造可变尺寸的自组装组织环使用定制的 3 d 印刷塑料模具。在 3 d-印刷模中固化了该底片;然后, 琼脂糖被投在固化的中和阴性。细胞被播种到产生的琼脂糖阱中, 它们聚集成组织环。

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 ...

fabrication-custom-agarose-wells-for-cell-seeding-tissue-ring-self

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

11.7K 次观看.  Worcester Polytechnic Institute. 该协议描述了一个平台, 用于制造可变尺寸的自组装组织环使用定制的 3 d 印刷塑料模具。在 3 d-印刷模中固化了该底片;然后, 琼脂糖被投在固化的中和阴性。细胞被播种到产生的琼脂糖阱中, 它们聚集成组织环。

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

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 ...

科研

生物工程

Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact bioprinting1-4 (extrusion, dip pen and soft lithography), contactless bioprinting5-7 (laser forward transfer, ink-jet deposition) and laser based techniques such as two photon photopolymerization8. It can be used for many applications such as tissue engineering9-13, biosensor microfabrication14-16 and as a tool to answer basic biological questions such as influences of co-culturing of different cell types17. Unlike common photolithographic or soft-lithographic methods, extrusion bioprinting has the advantage that it does not require a separate mask or stamp. Using CAD software, the design of the structure can quickly be changed and adjusted according to the requirements of the operator. This makes bioprinting more flexible than lithography-based approaches.
Here we demonstrate the printing of a sacrificial mold to create a multi-material 3D structure using an array of pillars within a hydrogel as an example. These pillars could represent hollow structures for a vascular network or the tubes within a nerve guide conduit. The material chosen for the sacrificial mold was poloxamer 407, a thermoresponsive polymer with excellent printing properties which is liquid at 4 &deg;C and a solid above its gelation temperature ~20 &deg;C for 24.5% w/v solutions18. This property allows the poloxamer-based sacrificial mold to be eluted on demand and has advantages over the slow dissolution of a solid material especially for narrow geometries. Poloxamer was printed on microscope glass slides to create the sacrificial mold. Agarose was pipetted into the mold and cooled until gelation. After elution of the poloxamer in ice cold water, the voids in the agarose mold were filled with alginate methacrylate spiked with FITC labeled fibrinogen. The filled voids were then cross-linked with UV and the construct was imaged with an epi-fluorescence microscope.

A bioprinter was used to create patterned hydrogels based on a sacrificial mold. The poloxamer mold was backfilled with a second hydrogel and then eluted, leaving voids which were filled with a third hydrogel. This method uses fast elution and good printability of poloxamer to generate complex architectures from biopolymers.

打印温敏反向模具图案创作的双组分水凝胶的三维细胞培养

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact ...

免疫与感染

Scaling of Engineered Vascular Grafts Using 3D Printed Guides and the Ring Stacking Method

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, engineered grafts offer great potential for patient treatment. However, engineered vascular grafts are generally not easily scalable, requiring manufacture of custom molds or polymer tubes in order to customize to different sizes, constituting a time-consuming and costly practice. Human arteries range in lumen diameter from about 2.0-38 mm and in wall thickness from about 0.5-2.5 mm. We have created a method, termed the "Ring Stacking Method," in which variable size rings of tissue of the desired cell type, demonstrated here with vascular smooth muscle cells (SMCs), can be created using guides of center posts to control lumen diameter and outer shells to dictate vessel wall thickness. These tissue rings are then stacked to create a tubular construct, mimicking the natural form of a blood vessel. The vessel length can be tailored by simply stacking the number of rings required to constitute the length needed. With our technique, tissues of tubular forms, similar to a blood vessel, can be readily manufactured in a variety of dimensions and lengths to meet the needs of the clinic and patient.

Scalable engineered blood vessels would improve clinical applicability. Using easily sizable 3D-printed guides, rings of vascular smooth muscle were created and stacked into a tubular form, forming a vascular graft. Grafts can be sized to meet the range of human coronary artery dimensions by simply changing the 3D-printed guide size.

工程血管移植缩放使用3D打印指南和环堆叠方法

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, ...

Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have more realistic biological responses that resemble the in vivo environment. Due to their advantages, tissue spheroids represent an emerging trend toward superior, more reliable, and more predictive study models with a broad range of biotechnological applicability. However, reproducible platforms that can achieve large-scale production of tissue spheroids have become an unmet need in fully exploring and boosting their potential. Herein, the large-scale production of homogeneous tissue spheroids is reported using a low-cost and time-effective methodology. A 3D printed stamp-like device is developed to generate up to 4,716 spheroids per 6-well plate. The device is fabricated by the stereolithography method using a photocurable resin. The final device is composed of cylindrical micropins, with a height of 1.3 mm and a width of 650 &#181;m. This approach allows the fast generation of homogeneous spheroids and co-cultured spheroids with uniform shape and size and &gt;95% cell viability. Moreover, the stamp-like device is tunable for different sizes of well plates and Petri dishes. It is easily sterilized and can be reused for long periods. The efficient large-scale production of homogeneous tissue spheroids is essential to leverage their translation for multiple areas of industry, such as tissue engineering, drug development, disease modeling, and on-demand personalized medicine.

<meta charset="utf8"></head><body>通过 3D 打印的邮票状设备生成组织球体

The present protocol describes a technique to produce tissue spheroids on a large scale cost-effectively using a 3D printed stamp-like device.

通过 3D 打印的邮票状设备生成组织球体

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have ...

生物化学

Custom Engineered Tissue Culture Molds from Laser-etched Masters

As the field of tissue engineering has continued to mature, there has been increased interest in a wide range of tissue parameters, including tissue shape. Manipulating tissue shape on the micrometer to centimeter scale can direct cell alignment, alter effective mechanical properties, and address limitations related to nutrient diffusion. In addition, the vessel in which a tissue is prepared can impart mechanical constraints on the tissue, resulting in stress fields that can further influence both the cell and matrix structure. Shaped tissues with highly reproducible dimensions also have utility for in vitro assays in which sample dimensions are critical, such as whole tissue mechanical analysis.
This manuscript describes an alternative fabrication method utilizing negative master molds prepared from laser etched acrylic: these molds perform well with polydimethylsiloxane (PDMS), permit designs with dimensions on the centimeter scale and feature sizes smaller than 25 &#181;m, and can be rapidly designed and fabricated at a low cost and with minimal expertise. The minimal time and cost requirements allow for laser etched molds to be rapidly iterated upon until an optimal design is determined, and to be easily adapted to suit any assay of interest, including those beyond the field of tissue engineering.

Herein we present a rapid, facile, and low-cost method for fabricating custom polydimethylsiloxane molds that can be used for producing hydrogel-based engineered tissues with complex geometries. We additionally describe results from mechanical and histological assessments conducted on engineered cardiac tissues produced using this technique.

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 ...

Initial 3D Cell Cluster Control in a Hybrid Gel Cube Device for Repeatable Pattern Formations

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its restrictions. Producing few repeatable results of pattern formation deteriorates the analysis of the mechanisms underlying the self-organization. Reducing variation in initial culture conditions, such as the cell density and distribution in the extracellular matrix (ECM), is crucial to enhance the repeatability of a 3D culture. In this article, we demonstrate a simple but robust procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain highly repeatable pattern formations. A micromold with a desired shape was fabricated by using photolithography or a machining process, and it formed a 3D pocket in the ECM contained in a hybrid gel cube (HGC). Highly concentrated cells were then injected in the pocket so that the cell cluster shape matched with the fabricated mold shape. The employed HGC allowed multi-directional scanning by its rotation, which enabled high-resolution imaging and the capture of the entire tissue structure even though a low-magnification lens was used. Normal human bronchial epithelial cells were used to demonstrate the methodology.&#160;

We present a procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain a repeatable pattern formation. A cubic device containing two different hydrogels is employed to achieve multi-directional imaging for tissue pattern formation.

用于可重复模式形成的混合凝胶立方体器件中的初始三维单元群集控制

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its ...

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

趋化梯度多细胞响应的三维分析

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and ...

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 ...

用于基于 FLIM 的代谢和缺氧分析的多细胞球状体形成

Production and Multi-Parameter Live Cell Fluorescence Lifetime Imaging Microscopy (FLIM) of Multicellular Spheroids

Multicellular tumor spheroids are a popular 3D tissue microaggregate&#160;model for reproducing tumor microenvironment, testing and optimizing drug therapies&#160;and using bio- and nanosensors in a 3D context. Their ease of production, predictable size, growth, and observed nutrient and metabolite gradients are important to recapitulate the 3D niche-like&#160;cell microenvironment. However, spheroid heterogeneity and variability of their production methods can influence overall cell metabolism, viability, and drug response. This makes it difficult to choose the most appropriate&#160;methodology, considering the requirements in size, variability, needs of biofabrication, and use as in vitro 3D tissue models in stem and cancer cell biology. In particular, spheroid production can influence their compatibility with quantitative live microscopies, such as optical metabolic imaging, fluorescence lifetime imaging microscopy (FLIM), monitoring of spheroid&#160;hypoxia with nanosensors, or viability. Here, a number of conventional spheroid formation protocols are presented, highlighting their compatibility with the live widefield, confocal, and two-photon microscopies. The follow-up imaging to analysis&#160;pipeline with multiplexed autofluorescence FLIM and, using various types of cancer and stem cell spheroids, is also presented.

作者聚焦:使用荧光寿命成像显微镜标准化用于代谢和氧合分析的球状体形成方法

Here, we describe different multicellular spheroid formation methods to perform follow-up multi-parameter live cell microscopy. Using fluorescence lifetime imaging microscopy (FLIM), cellular autofluorescence, staining dyes, and nanoparticles, the approach for analysis of cell metabolism, hypoxia, and cell death in live three-dimensional (3D) cancer and stem cell-derived spheroids is demonstrated.

多细胞球体的生产和多参数活细胞荧光寿命成像显微镜 （FLIM）

Multicellular tumor spheroids are a popular 3D tissue microaggregate&#160;model for reproducing tumor microenvironment, testing and optimizing drug ...

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

摘要

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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

打印温敏反向模具图案创作的双组分水凝胶的三维细胞培养

工程血管移植缩放使用3D打印指南和环堆叠方法

通过 3D 打印的邮票状设备生成组织球体

通过 3D 打印的邮票状设备生成组织球体

由激光蚀刻大师定制的组织培养模具

用于可重复模式形成的混合凝胶立方体器件中的初始三维单元群集控制

趋化梯度多细胞响应的三维分析

经济实惠的氧气显微镜辅助多细胞球体生物制造

多细胞球体的生产和多参数活细胞荧光寿命成像显微镜（FLIM）

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

摘要

探索更多视频

此视频中的章节

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

打印温敏反向模具图案创作的双组分水凝胶的三维细胞培养

工程血管移植缩放使用3D打印指南和环堆叠方法

通过 3D 打印的邮票状设备生成组织球体

通过 3D 打印的邮票状设备生成组织球体

由激光蚀刻大师定制的组织培养模具

用于可重复模式形成的混合凝胶立方体器件中的初始三维单元群集控制

趋化梯度多细胞响应的三维分析

经济实惠的氧气显微镜辅助多细胞球体生物制造

多细胞球体的生产和多参数活细胞荧光寿命成像显微镜 （FLIM）

多细胞球体的生产和多参数活细胞荧光寿命成像显微镜（FLIM）