Name: 3d printing bacteria to study motility and growth in complex 3d porous media
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Description: Watch this Scientific Journal Video about Author Spotlight: Studying Bacterial Growth in 3D Hydrogel Matrices at JoVE.com

R.K.B. 感谢总统博士后研究员计划的支持。本材料还基于 NSF 研究生研究奖学金计划资助 DGE-2039656（致 A.M.H.）支持的工作。A.S.D.-M.和 H.N.L. 感谢普林斯顿大学 Lidow 独立工作/高级论文基金的支持。我们还要感谢邦妮·巴斯勒（Bonnie Bassler）的实验室提供 霍乱弧菌菌株。S.S.D. 感谢 NSF 资助 CBET-1941716、DMR-2011750 和 EF-2124863 以及 Eric 和 Wendy Schmidt 变革技术基金、新泽西州健康基金会、皮尤生物医学学者计划和 Camille Dreyfus 教师学者计划的支持。

这种方法是使用 Tashman 等人先前建立的协议将商用 3D 熔融沉积建模打印机转换为 3D 生物打印机34。简而言之，Tashman等人用定制的注射泵挤出机取代了商用挤出机头。该挤出机能够以 3D 形式打印细菌细胞的高浓度液体悬浮液，其挤出体积和 3D 位置由 G 代码编程语言控制。挤出体积在软件中通过挤出机步骤（E-step）指定，并按照下文进一步说明进行校准。因此，这些细菌悬浮液被直接打印到颗粒状水凝胶基质中，该基质充当细胞的 3D 支持。下面，该协议还描述了如何制备具有不同聚合物浓度的基质，表征由此产生的孔径和流变特性的变化，并使用直接成像表征随后的细菌运动和生长。
1. 将商用3D打印机转换为3D生物打印机
<ol><li>从商用 3D 打印机上拆下挤出机和加热器（参见 材料表）。</li>
	<li>按照先前的协议制造注射泵挤出机34，并进行额外的修改以容纳一次性鲁尔锁注射器。将注射泵挤出机安装到打印机上。 注意:修改塑料注射器注射器泵挤出机所需的 CAD 文件在补充文件 1-3 中提供。</li>
	<li>在计算机上安装并打开 3D 打印机软件（请参阅 材料表）。将 3D 打印机连接到计算机。</li>
	<li>通过对齐机构的上半部分和下半部分，将带有适当尺寸针头的 1 mL 一次性注射器装入 3D 打印夹具中（图 1）。使用三个 M8 套筒螺栓和薄钢六角螺母将夹子固定在注射器周围（参见 材料表）。注射器柱塞与注射泵挤出机中的丝杠连接。通过旋转丝杠手动升高柱塞，以在注射器中产生 0.5 mL 的气隙。</li>
	<li>如果污染是实验的问题，请将注射器夹复合物运输到生物罩中，并在进入时用 70% 乙醇喷雾灭菌，然后再完成以下步骤。</li>
</ol>2.细菌悬浮液的制备
<ol><li>对于 霍乱弧菌和大肠杆菌， 在37°C的2%Lennox LB（Luria肉汤，参见 材料表）琼脂平板上生长过夜。<ol><li>对于 霍乱弧菌， 将细胞接种到 3 mL 液体 LB 中，并带有 10 个无菌玻璃珠。将细胞在37°C的振荡培养箱中生长5-6小时，直到指数中期至光密度（OD）600~0.9。</li>
		<li>对于 大肠杆菌， 将细胞接种到3mL液体LB中，在37°C的振荡培养箱中培养细胞过夜。将 200 μL 过夜培养物接种在新鲜 LB 中 3 小时，直至 OD 达到 0.6。</li>
	</ol></li>
	<li>将培养物转移到 10 mL 离心管中。在室温下以5,000× g 离心培养物5分钟以形成沉淀。除去上清液。用 ~10 μL 液体 LB 重悬，以达到 ~9 x 1010 个细胞/mL 的细胞密度。</li>
</ol>3. 将细菌悬浮液装入注射器中
注意:提供了两种将细菌加载到注射器中的方法（步骤3.1和步骤3.2）。步骤 3.1 用于加载少量细菌悬浮液 <200 μL，步骤 3.2 用于加载较大体积的细菌悬浮液 >200 μL。 步骤 3.1 用于此处所示的代表性结果。
<ol><li>将空的 1 mL 塑料鲁尔锁注射器装入 3D 生物打印机。将注射器柱塞与丝杠连接。手动缩回注射器以添加 0.2 mL 的气隙，为柱塞在注射器中移动提供空间，因为每批实验使用少量细胞 ~20-50 μL。<ol><li>将钝针头连接到注射器尖端，其针头尺寸与所需的打印特征尺寸相符。在这里，使用 2 英寸 20 G 针头。</li>
		<li>通过将细菌接种物置于针头下方的 10 mL 离心管，将细菌悬浮液加载到注射器中。手动旋转螺钉以缩回注射器柱塞并将细胞加载到注射器中。细菌细胞体积非常小，通常细胞只被加载到针头中。</li>
	</ol></li>
	<li>从注射器 - 夹复合物中取出柱塞，并使用另一个注射器和针头小心地将所需的细菌悬浮液加载到注射器 - 夹复合物中，小心避免捕获气泡。注射器 - 夹复合物应用所需的细菌悬浮液稍微填充边缘，然后转移到生物打印机上。<ol><li>小心地将没有柱塞的注射器夹复合物插入生物打印机挤出机主芯上的相应插座中。</li>
		<li>确保打印机托架大约在丝杠的一半处，并且装载的注射器下方有一个收集盘。然后，小心地将柱塞插入托架和注射器夹复合体，直到它卡在托架上。将柱塞缓慢压入细菌悬浮液中，以避免气泡滞留在注射器中。</li>
		<li>将适配器夹滑到柱塞背面的托架上，以将其固定到位，以便进行挤压和缩回操作。</li>
	</ol></li>
</ol>4. 挤出机步骤校准至沉积体积
<ol><li>要将挤出机步骤（E-step）校准到沉积体积，首先使用实验中使用的精确注射器，注射器针头和沉积细菌悬浮液设置生物打印机。在这里，使用 1 mL 鲁尔锁注射器。</li>
	<li>通过挤出任意 E 步长编号 （~200） 确定要校准的估计 E 步长范围，并使用注射器体积标记记录柱塞体积变化。</li>
	<li>使用此粗略的 E 步进与体积比来确定 E-step 设置以执行校准扫描。例如，如果 200 的 E 步长通过目视检查挤出约 20 μL，并且想要沉积 10-200 μL，则测试 100-2000 之间的 E-step。</li>
	<li>要进行线性校准扫描，首先在灵敏度为 0.1 mg 的分析天平上标记并测量 20 个 1.5 mL 采样管的干质量。</li>
	<li>将细菌悬浮液挤出到预先测量的 1.5 mL 管中。对于每个 E 步骤，至少执行 2 次重复。对线性范围内的所有 E 步重复，必要时更换细菌悬浮液。如果污染是实验的问题，则在每次样品后用浸有 70% 乙醇的无绒抹布擦拭注射器针头的外部。</li>
	<li>使用相同的分析天平测量所有 1.5 mL 试管的质量数。从第二个中减去第一个质量值，得到沉积的细菌悬浮液的净质量。</li>
	<li>将细菌悬浮液的质量转换为具有材料密度的体积。对于许多主要由水组成的细菌悬浮液，1 g/mL 是适当的密度近似值。</li>
	<li>在 E-step 和挤出体积之间执行线性配合以完成校准过程。</li>
</ol>5.颗粒状水凝胶基质的制备
<ol><li>在生物安全柜中，将交联丙烯酸/烷基丙烯酸酯共聚物的干燥颗粒（参见 材料表）加入 400 mL 2% Lennox Luria-Bertani （LB） 中，以保持基质无菌;然而，其他液体细胞培养基也可用于使水凝胶基质膨胀。 注意:添加到LB中的颗粒的重量百分比取决于目标孔径。在本研究中，对于0.9%颗粒状水凝胶基质，向LB中加入3.6 g干颗粒，对于1.2%颗粒状水凝胶基质，向LB中加入4.8 g干颗粒。通过在立式混合器中混合2分钟，将水凝胶颗粒均匀分散。</li>
	<li>混合后，通过添加 20 至 500 μL 增量的 10 M 氢氧化钠 （NaOH） 将 pH 调节至 7.4，以确保细胞活力。每次添加NaOH后，通过将移液管吸头浸入混合物中，然后将水凝胶基质擦拭到pH测试纸上来测量pH值。 注意:随着NaOH的加入，混合物的粘度会随着水凝胶颗粒开始膨胀而增加。膨胀的水凝胶颗粒直径为 ~5 μm 至 10 μm，并挤在水凝胶基质中。颗粒的内部网格尺寸为~40nm至100nm，如前所述32。网孔尺寸足够大，小分子（例如氧气和营养物质）可以自由扩散，但足够小，细菌可以限制在间隙孔之间。</li>
	<li>接下来，使用 50 mL 无菌塑料注射器将颗粒水凝胶基质转移到 50 mL 离心管中。在室温下以161× g 离心水凝胶基质1分钟，以除去混合过程中形成的气泡。</li>
	<li>让水凝胶基质在室温下静置至少 2 天，以确保没有发生污染。污染表现为悬浮在水凝胶基质中的微菌落。两天后，将水凝胶基质以161× g 离心1分钟，以除去形成的任何其他气泡。 注意:通过将水凝胶基质在室温下储存长达一周，可以在此处暂停该协议。</li>
	<li>在生物安全柜中，使用 30 mL 无菌塑料注射器，将所需量的水凝胶基质转移到将要进行打印的容器中（此处 ~20 mL 用于 20 mL 组织培养瓶或 1 mL 用于 1 mL 塑料微量比色皿）。</li>
</ol>6. 颗粒状水凝胶基质流变特性的表征
<ol><li>将 ~3 mL 的水凝胶基质加载到剪切流变仪（参见 材料表）中，粗糙的 50 mm 直径平行板之间留有 1 mm 的间隙，以测量流变特性。</li>
	<li>通过测量剪切应力作为剪切速率从 10-4 s-1 到 102 s-1 的对数扫描的函数，在剪切流变仪上使用单向剪切测量来量化屈服行为（例如， 图 2A）。 注意:在低剪切速率下，水凝胶基体将具有与剪切速率无关的恒定剪切应力（屈服应力）。在高剪切速率下，剪切应力将随着幂律对剪切速率的依赖而增加，表明水凝胶基体的流化。这种屈服-应力行为允许细菌在颗粒水凝胶基质内被3D打印29。</li>
	<li>使用应变幅度为 1% 且频率介于 0.1 至 1 Hz 之间的小振幅振荡流变学分别测量存储模量和损耗模量 G' 和 G'' 作为频率的函数（例如， 图 2B）。 注:用于3D打印的理想颗粒状水凝胶基质应具有大于损耗模量的存储模量，这表明介质充当卡住的弹性固体29。</li>
</ol>7. 颗粒状水凝胶基质间隙孔径的表征
<ol><li>将 100 nm 羧化荧光聚苯乙烯纳米颗粒（~3.6 x10 13 粒/mL，参见 材料表）在其包装中超声处理 15 分钟以重悬以分解任何聚集/颗粒簇。将 50 μL 纳米颗粒转移到 1.5 mL 微量离心管中。<ol><li>在室温下以9,500× g 离心10分钟，直到沉淀形成并且上清液澄清。除去上清液并将沉淀重悬于用于制备颗粒水凝胶基质的 1 mL 液体生长培养基（此处为 LB）中。 注意:通过将重悬的纳米颗粒在4°C下储存长达3个月，可以在此处暂停该协议。</li>
	</ol></li>
	<li>将重悬的纳米颗粒超声处理30分钟。将 1 mL 颗粒水凝胶基质转移到 1.5 mL 微量离心管中。将 1 μL 重悬的纳米颗粒加入颗粒状水凝胶基质中，并用移液器吸头混合。混合后，在室温下以161× g 离心30秒。</li>
	<li>将水凝胶基质和纳米颗粒混合物转移到直径为35mm的培养皿中，培养皿的玻璃底孔厚为0.1mm。该井的直径为20毫米，深度为1毫米。将玻璃盖玻片放在顶部并向下按压以禁用成像过程中的流动和蒸发。玻璃盖玻片的替代方法是在顶部加入 1 mL 石蜡油。</li>
	<li>使用共聚焦显微镜对纳米颗粒进行成像，该显微镜具有40倍油物镜，并在成像软件中增加了8倍的变焦（参见 材料表）。 注意:可以使用更高放大倍率的物镜，而不是在软件中使用额外的数码变焦。<ol><li>在单个 z 平面上对无延迟（理想情况下为 ~19 帧/秒）的时间循环进行 2 分钟的成像，视场内至少有四个纳米粒子。在不同位置重复 15 到 20 次，以收集足够的数据进行统计（100 到 200 个纳米颗粒）。</li>
	</ol></li>
	<li>使用粒子跟踪软件分析粒子的位移。在这里，使用基于经典 Crocker-Grier 算法的自定义脚本来跟踪纳米粒子35 的质心（参见 补充文件 7）。<ol><li>根据粒子跟踪，计算均方位移 （MSD）。MSD 将在短长度和时间尺度上表现出孔隙空间中的自由扩散，并且由于限制35，将在大长度和时间尺度上过渡到亚扩散缩放。</li>
	</ol></li>
	<li>确定向亚扩散缩放过渡的长度尺度，以估计局部孔径。通过将此长度刻度添加到纳米颗粒直径来计算孔径。对测量的每个纳米颗粒重复孔径分析。这将产生一个孔径分布，从中可以计算出平均孔径（例如， 图3）。</li>
</ol>8. 3D印刷工艺
<ol><li>用于样品容器的 3D 打印定制支架（CAD 文件参见 补充文件 4-6 ）。在这里，使用组织培养瓶和微量比色皿的支架。支架允许对打印机进行编程，以在一次打印会话中打印多个样品。将装有水凝胶培养基的样品容器放在构建平台上的支架中。</li>
	<li>打开3D打印软件。将预编程的 g 代码加载到软件中。对于具有代表性的结果，步骤3.1用于将细菌悬浮液加载到3D打印机中。 注: 表 1 给出了用于打印线性垂直几何形状的 g 代码示例。</li>
	<li>通过 3D 打印软件，移动 x-y-z 平面，使打印头在 x-y 平面上居中，成为第一个容器，然后归位 z 轴。归位 z 轴将抬起打印头。手动缓慢旋转螺钉以压下注射器柱塞，直到在针尖处可以看到少量细菌悬浮液。<ol><li>用无菌一次性湿巾轻轻擦去多余的细菌悬浮液。根据样品架、针头和注射器的高度，使用 3D 打印软件，将打印头降低到所选样品容器中的水凝胶介质中固定距离。单击"打印"开始打印过程。</li>
	</ol></li>
	<li>打印完成后，关闭样品容器。用 70% 乙醇擦拭打印机。妥善处理注射器和针头。</li>
</ol>9. 霍乱弧菌的生长和成像
<ol><li>对于大视场成像，请使用带有变焦镜头附件的相机，通过灯箱对细胞生长进行成像。在室温下打印后立即对样品进行成像，然后将它们转移到培养箱中。在实验期间，在成像会话之间将样品保持在固定培养箱中的37°C。</li>
	<li>在所需的时间内捕获图像，以观察颗粒状水凝胶基质中长时间的生长行为。</li>
</ol>

在水凝胶基质中生物打印细菌，用于研究运动和生长

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本出版物中用于3D打印和成像细菌群落的实验平台是普林斯顿大学代表Tapomoy Bhattacharjee和S.S.D.提交的专利申请的主题（PCT申请号PCT/US/2020/030213）。

协议中的关键步骤 重要的是要确保在制备每种水凝胶基质时，基质是在无菌环境中制备的。否则，可能会发生污染，例如，几天后基质中出现微菌落（小球体）。在混合过程中，重要的是所有干燥的颗粒状水凝胶颗粒都被溶解。此外，当用NaOH调节每个水凝胶基质的pH值时，颗粒会开始膨胀，这会增加水凝胶基质的粘度，导致混合更加困难。使用立式混合器将有助于确保NaOH充分混合到水凝胶基质中。在加载每种细菌悬浮液的过程中，针头中会形成气穴。为避免此问题，请确保针尖始终位于离心管的细菌悬浮液中，而不是在管的底部或靠近顶面。克服这个问题的另一种方法是培养大量细胞，从而具有更大体积的细菌悬浮液用于打印。
局限性 目前，在打印过程中，细菌悬浮液的低粘度限制了可以打印的几何形状，并且由于痕量细胞，通常会导致生物膜形成并在水凝胶基质表面的顶部生长。有几种潜在的方法可以克服这一限制，包括增加细菌悬浮液的粘度或进一步优化3D打印机设置。为了增加细菌悬浮液的粘度，可以将细菌悬浮液与另一种聚合物混合 - 例如，海藻酸盐，海藻酸盐之前已用于将细菌3D打印到平面上38。可以进一步优化打印机设置，以便在针头从颗粒水凝胶基质中取出时使注射器柱塞缩回，这有可能阻止细胞在从水凝胶基质中取出针头时沉积。
该方法相对于现有/替代方法的意义 这里描述的方法允许将细菌菌落打印成颗粒状水凝胶基质。颗粒状水凝胶基质可以研究外部环境因素（例如孔径、基质变形能力）对细菌运动和生长的影响。此外，虽然在这项工作中，LB被用作液体生长培养基来使水凝胶基质膨胀，但水凝胶基质可以被其他液体生长培养基（包括含有抗生素的培养基）肿胀。以前在密闭环境中研究细菌的方法受到实验时间长短、聚合物网格尺寸和周围水凝胶基质刚度的限制37,38。已经存在用不同聚合物制造颗粒状水凝胶基质的方案，因此研究不同环境条件对细菌运动和生长的影响的潜力是巨大的。这种方法允许在对照环境中研究细菌，这些环境更容易概括细菌在现实世界中栖息的环境，例如宿主粘液或土壤。许多其他方法的另一个局限性是周围基质的不透明度;然而，这种使用光学透明材料的方法提供了探索的能力，例如，光遗传学控制和3D细菌的图案化。
除了研究运动和生长之外，这里描述的3D打印方法克服了许多其他生物打印方法的局限性，这些方法需要将生物墨水沉积在基材上，因此，它们可以产生的工程生物材料的高度受到限制。未来，这种生物打印协议可以进一步扩展，通过将聚合物与生物膜形成细胞混合来制造生物杂化材料。与许多其他当前的细菌生物打印方法相比，颗粒状水凝胶基质为3D打印更厚，更大规模的工程生物材料和更复杂的几何形状提供支持。虽然这项工作只使用了 霍乱弧菌 和 大肠杆菌， 但其他物种，如 铜绿假单胞菌， 也成功地被3D打印37。除了打印之外，打印机还可以调整为在生长后对细菌进行受控采样，例如，查看是否有任何遗传变化。

细菌通常栖息在多样化、复杂的 3D 多孔环境中，从肠道和肺部的粘膜凝胶到地下的土壤1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、 16,17,18,19,20,21， 22,23,24,25.在这些环境中，细菌通过运动或生长的运动可能会受到周围障碍物的阻碍，例如聚合物网络或固体矿物颗粒的堆积物，从而影响细胞在其环境中扩散的能力26，获取营养来源，在新地形上定植，并形成保护性生物膜群落27.然而，传统的实验室研究通常采用高度简化的几何形状，专注于液体培养物或平面上的细胞。虽然这些方法对微生物学产生了关键的见解，但它们并没有完全概括自然栖息地的复杂性，导致与在现实世界环境中进行的测量相比，生长速度和运动行为存在巨大差异。因此，迫切需要一种方法来定义细菌菌落并研究它们在更类似于其许多自然栖息地的 3D 多孔环境中的运动和生长。
将细胞接种到琼脂凝胶中，然后通过肉眼或使用相机观察它们的宏观扩散，提供了一种实现此目的的直接方法，正如 Tittsler 和 Sandholzer 于 1936年首次提出的那样 28.然而，这种方法存在许多关键的技术挑战:（1）虽然原则上可以通过改变琼脂糖浓度来改变孔径，但这种凝胶的孔结构定义不清;（2）光散射导致这些凝胶浑浊，难以以高分辨率和高保真度在单个尺度上可视化细胞，特别是在大样品中;（3）当琼脂浓度过大时，细胞迁移仅限于凝胶的顶部平坦表面;（4） 这种凝胶的复杂流变性使得引入具有明确几何形状的接种物具有挑战性。
为了解决这些局限性，在之前的工作中，Datta的实验室开发了一种替代方法，使用颗粒状水凝胶基质 - 由在液体细菌培养物中膨胀的卡住的生物相容性水凝胶颗粒组成 - 作为"多孔培养皿"来限制3D细胞。这些基质是柔软的、自愈的屈服应力固体;因此，与用于其他生物打印工艺的交联凝胶不同，注射微喷嘴可以通过局部重新排列水凝胶颗粒29，沿着任何规定的3D路径在基质内自由移动。然后，这些颗粒迅速重新致密化并在注射的细菌周围自我修复，从而在没有任何额外有害处理的情况下将细胞支撑到位。因此，该过程是3D打印的一种形式，它使细菌细胞能够以所需的3D结构排列，具有确定的群落组成，位于具有可调物理化学性质的多孔基质中。此外，水凝胶基质是完全透明的，可以使用成像直接可视化细胞。
这种方法的实用性之前已经通过两种方式得到了证明。在一组研究中，稀释的细胞分散在整个水凝胶基质中，这使得研究单个细菌的运动性成为可能30,31。在另一组研究中，使用安装在可编程显微镜载物台上的注射喷嘴在厘米级凝胶中3D打印多细胞群落，这使得研究细菌群落在其周围环境中的传播成为可能32,33。在这两种情况下，这些研究都揭示了与液体培养/平面上的细菌相比，栖息在多孔环境中的细菌的传播特性存在以前未知的差异。然而，鉴于它们被安装在显微镜载物台上，这些先前的研究仅限于小样品体积（~1 mL），因此实验时间尺度很短。它们在定义具有高空间分辨率的接种物几何形状方面的能力也受到限制。
在这里，描述了解决这两个限制的下一代实验平台。具体来说，提供了协议，通过该协议，人们可以使用带有注射器挤出机的改进的3D打印机进行3D打印并大规模成像细菌菌落。此外，代表性数据表明，以生物膜形成 霍乱弧 菌和浮游 大肠杆 菌为例，这种方法可用于研究细菌的运动和生长。这种方法使细菌菌落能够长时间维持，并使用各种成像技术进行可视化。因此，这种方法在3D多孔生境中研究细菌群落的能力具有巨大的研究和应用潜力，影响了肠道、皮肤、肺和土壤中微生物的治疗和研究。此外，这种方法将来可用于将基于细菌的工程生物材料3D打印成更复杂的独立形状。

<table><tbody><tr><td>1 mL cuvettes</td><td>VWR</td><td>97000-586</td><td></td></tr><tr><td>1 mL Luer lock syringe</td><td>BH Supplies</td><td>BH1LL</td><td></td></tr><tr><td>10 M NaOH</td><td>Sigma-Aldrich</td><td>72068</td><td></td></tr><tr><td>100 nm carboxylated fluorescent polystyrene nanoparticles (FluoSpheres)&amp;nbsp;</td><td>&amp;nbsp;Invitrogen, (ThermoFischer Scientific)</td><td>&lt;br/&gt; F8803</td><td></td></tr><tr><td>15 mL centrifuge tubes</td><td>ThermoFischer Scientific</td><td>14-955-237</td><td></td></tr><tr><td>20 G blunt needle</td><td>McMaster Carr</td><td>75165A252</td><td></td></tr><tr><td>25 mL tissue culture flasks</td><td>VWR</td><td>10861-566</td><td></td></tr><tr><td>3D printer</td><td>Lulzbot</td><td>LulzBot Mini 2</td><td></td></tr><tr><td>3D printing software</td><td>Cura</td><td>Cura-Lulzbot</td><td></td></tr><tr><td>50 mL centrifuge tubes</td><td>ThermoFischer Scientific</td><td>14-955-239</td><td></td></tr><tr><td>Agar</td><td>Sigma-Aldrich</td><td>A1296</td><td></td></tr><tr><td>Carbomer Granular Hydrogel Particles</td><td>Lubrizol&amp;nbsp;</td><td>Carbopol 980NF</td><td>dry granules of crosslinked acrylic acid/alkyl acrylate copolymers</td></tr><tr><td>Centrifuge (2 mL tube capacity)</td><td>VWR</td><td>2405-37</td><td></td></tr><tr><td>Centrifuge (50 mL tube capacity)</td><td>ThermoFischer Scientific</td><td>75007200</td><td>Sorvall (brand) ST 8 (model)</td></tr><tr><td>Confocal Microscope</td><td>Nikon</td><td>A1R+ inverted laserscanning&lt;br/&gt; confocal microscope</td><td></td></tr><tr><td>Glass bottom petri dish</td><td>Cellvis</td><td>D35-10-1-N</td><td></td></tr><tr><td>Lennox LB (Lubria Broth)</td><td>Sigma-Aldrich</td><td>L3022</td><td></td></tr><tr><td>M8 &amp;times; 1.25 mm, 150 mm long, Fully Threaded Socket Cap</td><td>McMaster Carr</td><td>91290A478</td><td></td></tr><tr><td>M8 &amp;times; 1.25 mm, Brass Thin Hex Nut</td><td>McMaster Carr</td><td>93187A300</td><td></td></tr><tr><td>Open-source syringe pump</td><td>Custom-made</td><td>Replistruder 4</td><td>https://www.sciencedirect.com/science/article/pii/S2468067220300791</td></tr><tr><td>Petri dish (60 mm round)</td><td>ThermoFischer Scientific</td><td>FB0875713A</td><td></td></tr><tr><td>Shear Rheometer</td><td>Anton Paar</td><td>MCR 501</td><td></td></tr><tr><td>Ultrasonic cleaner</td><td>VWR</td><td>97043-992</td><td></td></tr></tbody></table>

3d printing bacteria to study motility and growth in complex 3d porous media

细菌在复杂的三维 （3D） 多孔环境中无处不在，例如生物组织和凝胶，以及地下土壤和沉积物。然而，以前的大多数工作都集中在对散装液体或平坦表面的细胞的研究上，这并不能完全概括许多天然细菌栖息地的复杂性。在这里，通过描述一种将致密的细菌菌落3D打印成卡住的颗粒状水凝胶基质的方法的发展，解决了这一知识空白。这些基质具有可调的孔径和机械性能;它们在物理上限制了细胞，从而在3D中支持它们。它们是光学透明的，允许使用成像直接可视化细菌在周围环境中的传播。作为这一原理的证明，在这里，通过3D打印和成像非运动和运动 性霍乱弧菌以及非运动性 大肠杆菌， 在具有不同间质孔径的卡住颗粒水凝胶基质中证明了该协议的能力。

Watch this Scientific Journal Video about Author Spotlight: Studying Bacterial Growth in 3D Hydrogel Matrices at JoVE.com

作者聚焦:研究 3D 水凝胶基质中的细菌生长

该协议描述了细菌菌落的三维（3D）打印程序，以研究它们在复杂的3D多孔水凝胶基质中的运动和生长，这些基质比传统的液体培养物或培养皿更类似于其自然栖息地。

3D打印细菌研究复杂3D多孔介质中的运动和生长

Bacteria are ubiquitous in complex three-dimensional (3D) porous environments, such as biological tissues and gels, and subsurface soils and sediments. ...

细菌在复杂的三维多孔环境中无处不在，例如生物组织和凝胶以及地下土壤和沉积物。在这里，我们开发了一种将密集的细菌菌落3D打印到卡住的颗粒状水凝胶基质中的方法，以研究它们在复杂环境中的生长和运动。研究表明，与在平坦表面上的液体培养物相比，栖息在多孔环境中的细菌的传播特性存在以前未知的差异。开发使用颗粒状水凝胶基质，该基质由在液体细菌培养物中膨胀的卡住的生物相容性水凝胶颗粒组成，作为多孔培养皿来限制3D细胞。以前的研究仅限于约一毫升的小样品体积，因此实验时间尺度短，并且在定义具有高空间分辨率的接种几何形状的能力方面也受到限制。我们已经确定细菌的细胞扩散特性取决于细胞的孔径和运动性。

3d-printing-bacteria-to-study-motility-growth-complex-3d-porous

Research

JoVE Journal

Bioengineering

3D Printing Bacteria to Study Motility and Growth in Complex 3D Porous Media

2.0K 次观看.  University of Colorado Boulder. 细菌在复杂的三维多孔环境中无处不在，例如生物组织和凝胶以及地下土壤和沉积物。在这里，我们开发了一种将密集的细菌菌落3D打印到卡住的颗粒状水凝胶基质中的方法，以研究它们在复杂环境中的生长和运动。研究表明，与在平坦表面上的液体培养物相比，栖息在多孔环境中的细菌的传播特性存在以前未知的差异。开发使用颗粒状水凝胶基质，该基质由在液体细?...

视频: 作者聚焦:研究 3D 水凝胶基质中的细菌生长

From Voxels to Knowledge: A Practical Guide to the Segmentation of Complex Electron Microscopy 3D-Data

Modern 3D electron microscopy approaches have recently allowed unprecedented insight into the 3D ultrastructural organization of cells and tissues, enabling the visualization of large macromolecular machines, such as adhesion complexes, as well as higher-order structures, such as the cytoskeleton and cellular organelles in their respective cell and tissue context. Given the inherent complexity of cellular volumes, it is essential to first extract the features of interest in order to allow visualization, quantification, and therefore comprehension of their 3D organization. Each data set is defined by distinct characteristics, e.g., signal-to-noise ratio, crispness (sharpness) of the data, heterogeneity of its features, crowdedness of features, presence or absence of characteristic shapes that allow for easy identification, and the percentage of the entire volume that a specific region of interest occupies. All these characteristics need to be considered when deciding on which approach to take for segmentation.
The six different 3D ultrastructural data sets presented were obtained by three different imaging approaches: resin embedded stained electron tomography, focused ion beam- and serial block face- scanning electron microscopy (FIB-SEM, SBF-SEM) of mildly stained and heavily stained samples, respectively. For these data sets, four different segmentation approaches have been applied: (1) fully manual model building followed solely by visualization of the model, (2) manual tracing segmentation of the data followed by surface rendering, (3) semi-automated approaches followed by surface rendering, or (4) automated custom-designed segmentation algorithms followed by surface rendering and quantitative analysis. Depending on the combination of data set characteristics, it was found that typically one of these four categorical approaches outperforms the others, but depending on the exact sequence of criteria, more than one approach may be successful. Based on these data, we propose a triage scheme that categorizes both objective data set characteristics and subjective personal criteria for the analysis of the different data sets.

The bottleneck for cellular 3D electron microscopy is feature extraction (segmentation) in highly complex 3D density maps. We have developed a set of criteria, which provides guidance regarding which segmentation approach (manual, semi-automated, or automated) is best suited for different data types, thus providing a starting point for effective segmentation.

从体素知识:实用指南复电镜三维数据的分割

Modern 3D electron microscopy approaches have recently allowed unprecedented insight into the 3D ultrastructural organization of cells and tissues, ...

科研

生物工程

Combining Fluidic Devices with Microscopy and Flow Cytometry to Study Microbial Transport in Porous Media Across Spatial Scales

Understanding the transport, dispersion and deposition of microorganisms in porous media is a complex scientific task comprising topics as diverse as hydrodynamics, ecology and environmental engineering. Modeling bacterial transport in porous environments at different spatial scales is critical to better predict the consequences of bacterial transport, yet current models often fail to up-scale from laboratory to field conditions. Here, we introduce experimental tools to study bacterial transport in porous media at two spatial scales. The aim of these tools is to obtain macroscopic observables (such as breakthrough curves or deposition profiles) of bacteria injected into transparent porous matrices. At the small scale (10-1000 &#181;m), microfluidic devices are combined with optical video-microscopy and image processing to obtain breakthrough curves and, at the same time, to track individual bacterial cells at the pore scale. At larger scale, flow cytometry is combined with a self-made robotic dispenser to obtain breakthrough curves. We illustrate the utility of these tools to better understand how bacteria are transported in complex porous media such as the hyporheic zone of streams. As these tools provide simultaneous measurements across scales, they pave the way for mechanism-based models, critically important for upscaling. Application of these tools may not only contribute to the development of novel bioremediation applications but also shed new light on the ecological strategies of microorganisms colonizing porous substrates.

Breakthrough curves (BTCs) are efficient tools to study the transport of bacteria in porous media. Here we introduce tools based on fluidic devices in combination with microscopy and flow cytometric counting to obtain BTCs.

将流体设备与显微镜和流式细胞学相结合，研究跨空间尺度的多孔介质中的微生物传输

Understanding the transport, dispersion and deposition of microorganisms in porous media is a complex scientific task comprising topics as diverse as ...

环境科学

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D胶原凝胶和微通道3D相互作用的研究准备<em&gt;在体内</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

2D and 3D Matrices to Study Linear Invadosome Formation and Activity

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor cells have to cross anatomical barriers to invade and migrate through the surrounding tissue in order to reach blood or lymphatic vessels. This requires the interaction between cells and the extracellular matrix (ECM). At the cellular level, many cells, including the majority of cancer cells, are able to form invadosomes, which are F-actin-based structures capable of degrading ECM. Invadosomes are protrusive actin structures that recruit and activate matrix metalloproteinases (MMPs). The molecular composition, density, organization, and stiffness of the ECM are crucial in regulating invadosome formation and activation. In vitro, a gelatin assay is the standard assay used to observe and quantify invadosome degradation activity. However, gelatin, which is denatured collagen I, is not a physiological matrix element. A novel assay using type I collagen fibrils was developed and used to demonstrate that this physiological matrix is a potent inducer of invadosomes. Invadosomes that form along the collagen fibrils are known as linear invadosomes due to their linear organization on the fibers. Moreover, molecular analysis of linear invadosomes showed that the discoidin domain receptor 1 (DDR1) is the receptor involved in their formation. These data clearly demonstrate the importance of using a physiologically relevant matrix in order to understand the complex interactions between cells and the ECM.

This protocol describes how to prepare a 2D mixed matrix, consisting of gelatin and collagen I, and a 3D collagen I plug to study linear invadosomes. These protocols allow for the study of linear invadosome formation, matrix degradation activity, and the invasion capabilities of primary cells and cancer cell lines.

2D和3D矩阵研究线性侵袭体形成和活性

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor ...

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

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

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

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

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

Novel Process for 3D Printing Decellularized Matrices

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can provide mechanical stability similar to native tissue while biologic agents offer compositional cues to progenitor cells, leading to their migration, proliferation, and differentiation to reconstitute the original tissues/organs1,2. Unfortunately, many 3D printing compatible, bioresorbable polymers (such as polylactic acid, PLA) are printed at temperatures of 210 &#176;C or higher - temperatures that are detrimental to biologics. On the other hand, polycaprolactone (PCL), a different type of polyester, is a bioresorbable, 3D printable material that has a gentler printing temperature of 65 &#176;C. Therefore, it was hypothesized that decellularized extracellular matrix (DM) contained within a thermally protective PLA barrier could be printed within PCL filament and remain in its functional conformation. In this work, osteochondral repair was the application for which the hypothesis was tested. As such, porcine cartilage was decellularized and encapsulated in polylactic acid (PLA) microspheres which were then extruded with polycaprolactone (PCL) into filament to produce 3D constructs via fused deposition modeling. The constructs with or without the microspheres (PLA-DM/PCL and PCL(-), respectively) were evaluated for differences in surface features.

This protocol describes the production of polycaprolactone (PCL) filament with embedded polylactic acid (PLA) microspheres which contain decellularized matrices (DM) for 3D printing of structural tissue engineering constructs.

一种新的三维打印解光矩阵处理方法

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can ...

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 ...

Three-dimensional Patterning of Engineered Biofilms with a Do-it-yourself Bioprinter

Biofilms are aggregates of bacteria embedded in a self-produced spatially-patterned extracellular matrix. Bacteria within a biofilm develop enhanced antibiotic resistance, which poses potential health dangers, but can also be beneficial for environmental applications such as purification of drinking water. The further development of anti-bacterial therapeutics and biofilm-inspired applications will require the development of reproducible, engineerable methods for biofilm creation. Recently, a novel method of biofilm preparation using a modified three-dimensional (3D) printer with a bacterial ink has been developed. This article describes the steps necessary to build this efficient, low-cost 3D bioprinter that offers multiple applications in bacterially-induced materials processing. The protocol begins with an adapted commercial 3D printer in which the extruder has been replaced with a bio-ink dispenser connected to a syringe pump system enabling a controllable, continuous flow of bio-ink. To develop a bio-ink suitable for biofilm printing, engineered Escherichia coli bacteria were suspended in a solution of alginate, so that they solidify in contact with a surface containing calcium. The inclusion of an inducer chemical within the printing substrate drives expression of biofilm proteins within the printed bio-ink. This method enables 3D printing of various spatial patterns composed of discrete layers of printed biofilms. Such spatially-controlled biofilms can serve as model systems and can find applications in multiple fields that have a wide-ranging impact on society, including antibiotic resistance prevention or drinking water purification, among others.

This article describes a method of transforming a low-cost commercial 3D printer into a bacterial 3D printer that can facilitate printing of patterned biofilms. All necessary aspects of preparing the bioprinter and bio-ink are described, as well as verification methods to assess the formation of biofilms.

工程生物膜的三维造型

Biofilms are aggregates of bacteria embedded in a self-produced spatially-patterned extracellular matrix. Bacteria within a biofilm develop enhanced ...

Generating Controlled, Dynamic Chemical Landscapes to Study Microbial Behavior

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the microscale&#8213;to create micro-environments for microbial chemotaxis experiments. To create chemical pulses, we developed a system to introduce amino acid sources near-instantaneously by photolysis of caged amino acids within a polydimethylsiloxane (PDMS) microfluidic chamber containing a bacterial suspension. We applied this method to the chemotactic bacterium, Vibrio ordalii, which can actively climb these dynamic chemical gradients while being tracked by video microscopy. Amino acids, rendered biologically inert (&#39;caged&#39;) by chemical modification with a photoremovable protecting group, are uniformly present in the suspension but not available for consumption until their sudden release, which occurs at user-defined points in time and space by means of a near-UV-A focused LED beam. The number of molecules released in the pulse can be determined by a calibration relationship between exposure time and uncaging fraction, where the absorption spectrum after photolysis is characterized by using UV-Vis spectroscopy. A nanoporous polycarbonate (PCTE) membrane can be integrated into the microfluidic device to allow the continuous removal by flow of the uncaged compounds and the spent media. A strong, irreversible bond between the PCTE membrane and the PDMS microfluidic structure is achieved by coating the membrane with a solution of 3-aminopropyltriethoxysilane (APTES) followed by plasma activation of the surfaces to be bonded. A computer-controlled system can generate user-defined sequences of pulses at different locations and with different intensities, so as to create resource landscapes with prescribed spatial and temporal variability. In each chemical landscape, the dynamics of bacterial movement at the individual scale and their accumulation at the population level can be obtained, thereby allowing the quantification of chemotactic performance and its effects on bacterial aggregations in ecologically relevant environments.

A protocol for the generation of dynamic chemical landscapes by photolysis within microfluidic and millifluidic setups is presented. This methodology is suitable to study diverse biological processes, including the motile behavior, nutrient uptake, or adaptation to chemicals of microorganisms, both at the single cell and population level.

生成受控动态化学景观，研究微生物行为

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the ...

Measuring the Migration and Biofilm Formation of Various Bacteria

As microbes that thrive in the host body primarily have adaptive abilities that facilitate their survival, methods for classifying and identifying their nature would be beneficial in facilitating their characterization. Currently, most studies focus only on one specific characterization method; however, the isolation and identification of microorganisms from the host is a continuous process and usually requires several combinatorial characterization methods. Herein, we describe methods of identifying the microbial biofilm-forming ability, the microbial respiration state, and their chemotaxis behavior. The methods are used to identify five microbes, three of which were isolated from the bone tissue of Sprague-Dawley (SD) rats (Corynebacterium stationis, Staphylococcus cohnii subsp. urealyticus, and Enterococcus faecalis) and two from the American Type Culture Collection (ATCC)-Staphylococcus aureus ATCC 25923&#160;and Enterococcus faecalis V583. The microbes isolated from the SD rat bone tissue include the gram-positive microbes. These microbes have adapted to thrive under stressful and nutrient-limiting environments within the bone matrix. This article aims to provide the readers with the specific know-how of determining the nature and behavior of the isolated microbes within a laboratory setting.

Here, we present a practical method for the isolation and identification of microorganisms within the host. In this way, the physicochemical properties of microorganisms and possible ways of living in the host are clearly described.

As microbes that thrive in the host body primarily have adaptive abilities that facilitate their survival, methods for classifying and identifying their ...