这项工作得到了澳大利亚高级分子成像研究中心（CE140100011）（http://www.imagingcoe.org/）的支持。这项研究部分使用澳大利亚同步加速器的MX2光束线进行，这是ANSTO的一部分，并利用了澳大利亚癌症研究基金会（ACRF）探测器。

1. 使用玻璃注射器在高粘性介质中制备晶体
<ol><li>轻轻地将水晶溶液（约1，000 x g，+10分钟，22°C）中离心，形成柔软的晶体颗粒并去除多余的缓冲区。 这将导致颗粒中的晶体高度浓度，可用于数据收集。 注：为防止粘性介质稀释，在此步骤中增加晶体浓度。优化粘性介质与晶体体积的比例，以获得高浓度的晶体，同时保持媒体的高粘度。离心晶体溶液形成颗粒，并去除多余的缓冲区，以增加溶液中的晶体浓度，如 Darmanin 等所述。阿尔. 29.</li>
	<li>用耦合器设置两个 100μL 注射器。</li>
	<li>将耦合器固定到第一个注射器的末端，并在注射器顶部添加 28 μL 的水晶溶液。慢慢地，将柱塞插入注射器顶部，轻轻地将溶液向下推至耦合器尖端，去除形成的任何气泡。遵循下面讨论的两种方法之一来做到这一点。 注：如果晶体不显著改变整体样品的粘度，则添加到高粘度介质中的晶体体积可能会有所不同。<ol>
<li>第一种方法：使用快速压力变化来爆裂气泡。<ol>
<li>小心地将一个戴手套的手指放在钝化耦合针尖的顶部，然后（非常轻轻地）施加压力以创建密封件。不要施加太大的压力，因为这可能会导致针棒受伤。</li>
			<li>现在将柱塞拉回来，将解决方案从针头点拉开，这将在注射器中产生积压。</li>
			<li>通过从钝化的针尖上取下戴手套的手指，快速释放注射器顶部的压力。请小心，如果取出手指时样品离尖端太近，可能会喷出，导致样品丢失。注射器内压力的快速变化会破裂气泡。重复，直到所有气泡都被移除。</li>
		</ol>
</li>
		<li>第二种方法：使用"人离心机"去除气泡。<ol>
<li>将注射器放在一只手上，针头朝上，柱塞夹在两根手指之间，使其无法移动。</li>
			<li>快速将手持注射器的手臂向一个方向旋转 2-3 次，结果样品上的离心力将迫使任何气泡从注射器中流出。小心点，如果做得太慢，样品可能会丢失。</li>
			<li>检查注射器是否存在气泡，如果气泡仍然存在，重复步骤 1.3.2.1 – 1.3.2.2，直到清除所有气泡。</li>
		</ol>
</li>
	</ol></li>
	<li>使用细铲，将 +42 μL 的高真空硅胶油脂直接添加到第二个注射器的顶部。将柱塞一直推至末端，清除所有气泡，并确保注射器末端没有气隙。 注：硅胶油脂的精确成分并不关键，因为它充当惰性载体。粘度值 =gt;10 Pa.s 或大约 60：40 硅胶油脂与晶体溶液的比例是最佳注射方式，但是，根据样品的确切特征，这种情况可能有所不同。调整添加到硅胶油脂中的晶体溶液体积，以优化混合物中的晶体浓度，如讨论中所述。硅胶润滑脂以外的高粘度介质只要与样品30、31、32、33兼容即可使用。</li>
	<li>确保两个注射器中的任何一个都没有气泡，并使用耦合器将注射器连接在一起。垂直握住注射器，只需抓住注射器的末端，因为由于手指产生的热量而使注射器变热会影响样品。</li>
	<li>将样品混合在一起，轻轻压低晶体溶液侧的柱塞，使其混合到硅胶油脂中，然后将柱塞推入硅胶油脂侧，从而将样品推回晶体溶液侧。轻轻重复这个过程50至100次，使样品彻底混合，看起来均匀。 注：如果晶体直接生长在高粘性介质中（例如脂质立方相（LCP），则不需要步骤 1.1-1.6。在刘 et.al 中可以找到注射器内生长晶体的协议。34，35。按照此协议进行样品整合，步骤 10，其中所有样本现在都包含在一个注射器中，并删除结晶缓冲区。本协议不需要添加 7.9MAG。相反，通过轻轻将柱塞推下样品，直到注射器中不再保留液体，从注射器中取出所有多余的液体。</li>
	<li>通过注射器在光学显微镜下可视化晶体，或者为了获得最佳效果，将少量 （+1 μL） 提取到玻璃滑梯上，并在顶部放置盖滑。</li>
	<li>检查晶体浓度。<ol>
<li>通过使用光学显微镜图像计算特定区域的晶体数量，确定硅胶油脂中的晶体浓度。为了获得最佳效果，在整个介质中均匀分配的高密度晶体是理想的（+106 晶体/mL），以获得较高的晶体命中率。</li>
		<li>通过在步骤 1.3 和 1.4 中更改晶体与粘性介质的比例来调整注射器中的晶体浓度。<ol>
<li>为了降低晶体浓度，请在第 1.4 步增加硅胶润滑脂/HV 介质的量，或在步骤 1.1 中设置注射器之前用结晶缓冲器稀释溶液中的晶体颗粒，从而稀释注射器中的晶体。</li>
			<li>要增加晶体浓度，请在第 1.4 步减少硅胶油脂的含量，但请注意不要将粘度降低到 10 Pa.s 以下。 注：此处报告的晶体浓度针对此特定样本和晶体尺寸进行了优化。然而，理想的晶体浓度将取决于晶体大小、光束大小、针头内部直径（I.D.）和事件X射线通量。这可以从命中率中确定，最佳命中率为 ~30%，被视为"良好"。在实践中，必须在光束时间针对不同的样品优化晶体浓度，以获得所需的命中率。从高浓度晶体开始，109 个晶体/mL。调整晶体浓度的程序在刘 et.al。35.</li>
		</ol>
</li>
		<li>协议可在此处暂停，直到喷油器准备好进行数据收集。<ol>
<li>如果晶体生长在LCP中，则密封它们，让它们在室温下在注射器中停留几天。</li>
			<li>如果晶体已转移到不同的高粘度介质（例如硅胶润滑脂），则应在测量前对晶体进行稳定性测试。这将确定晶体在惰性介质中稳定多长时间（理想情况下>8 h）和数据收集的时间限制。在这里，溶酶晶体在硅胶油脂中稳定了数天，使用光学显微镜检查时没有溶解的迹象。</li>
		</ol>
</li>
	</ol></li>
	<li>在断开耦合器之前，将所有样品移动到一个注射器中。将耦合器与注射器断开并连接注射针。将针头牢牢拧入注射器底部，确保紧固针头以防止泄漏。这个实验使用了108μm I.D.针（针长13毫米，点式3）。根据晶体大小和光束大小，连接 51μm 或 108μm I.D. 针头。 注：需要在光束时间之前对样品流进行预先测试，才能选择正确的喷油器针尺寸。根据样品特性（即粘度、充电、缓冲成分）和针头 I.D.，样品的流动方式会有所不同。因此，建议测试各种针头尺寸，以尽可能小的 I.D. 针产生最稳定的流，以最大限度地提高数据收集过程中的信号噪声比。</li>
	<li>如果喷油器已经安装并对齐在光束线上，则移动到第 3 节，并将样品注射器直接安装到喷油器上。 注：协议可在此处暂停。</li>
</ol>2. 喷油器安装和控制
<ol><li>将喷油器安装到光束线上。取出 MX2 光束线上的低温喷嘴，然后用喷油器更换。松开固定低温喷嘴的夹紧螺钉，并将其连接到位于电动舞台旁边的支架上。</li>
	<li>将喷油器从手推车上抬起来，握住黑色手柄，放在电动舞台上。<ol>
<li>将喷油器固定在机动阶段：手拧紧黑色夹紧螺丝旋钮。</li>
	</ol>
</li>
	<li>将空注射器安装到喷油器上<ol>
<li>在喷油器计算机控制接口（即 EPOS 位置控制软件）中，在软件菜单中选择工具下的Homing 模式。然后选择负限制开关，并开始霍明，让软件运行，直到螺丝被收回足够的注射器，以适应螺丝下。如果离开运行无人监督的限制开关自动停止螺钉。</li>
		<li>通过注射器支架中的缝隙插入针头，将空（"dummy"）注射器安装到喷油器上。将注射器与支架对接。</li>
		<li>用两个O环固定注射器。<ol>
<li>将第一个 O 形环包裹在注射器的中间部分，将其连接到注射器两侧的钩子上。</li>
			<li>将第二个 O 形环绕在注射器上部的挂钩上，然后将 O 环的一部分放在玻璃注射器的顶部，如演示和图 1B所示。</li>
		</ol>
</li>
	</ol>
</li>
	<li>将喷油器与 X 射线束对齐<ol>
<li>通过光束线控制软件，将喷油器的机动阶段移动到 X 射线交互点。这可以使用内联相机进行可视化。光束的大小和位置由屏幕上的红十字表示，可移动电动舞台，使针头与 X 射线交互区域对齐。</li>
		<li>调整阶段的 x 和 y 位置，使针头与红十字对齐。</li>
		<li>调整 z 位置，直到针尖对焦。</li>
		<li>通过检查注射器尖端是否与光束线相机生成的光学显微镜图像中可见的十字线相符，可直观地验证针尖和 X 射线光束的对齐。</li>
		<li>针尖对齐后，将尖端移到交叉毛发上方~100μm。这消除了针尖的 X 射线散射。 注：同步加速器MX2光束线上的喷油器的安装和对齐必须由光束线科学家进行，并且可以在30分钟内完成。</li>
	</ol>
</li>
</ol>3. 安装样品注射器
<ol><li>按照步骤 2.3 将空注射器替换为样品注射器。</li>
	<li>将喷油器盖放在样品注射器柱塞头上。</li>
	<li>将驱动螺钉移到盖的顶部。</li>
	<li>在喷油器控制软件中选择 速度模式。</li>
	<li>输入 3000 rpm （+115 nL/s） 作为 设置值 并按 应用设置值。</li>
	<li>当驱动螺丝碰到注射器柱塞头的盖时，请停止电机。<ol>
<li>将喷油器控制软件中的 rpm 值设置为零，当螺钉接近盖子时， 将设置值 更改为"0"。</li>
		<li>接触后，通过按 应用设置值 立即停止螺钉来激活预设值。</li>
	</ol>
</li>
	<li>轻轻摇动盖子，确保盖牢牢地握住。 注：每当在控制软件中的 "设置值 "框中输入新的集值时，它只有在按下 "应用设置值"后才会激活。</li>
</ol>4. 运行喷油器
<ol><li>盖驱动器螺钉与柱塞顶部牢固接触后，将转速 设置值 更改为 100。这相当于~4 nL/s。</li>
	<li>当样品首次出来时，可直观地检查针尖，或查看针头的光束线相机图像，以观察样本何时开始从针尖挤出。</li>
	<li>搜索小屋，关闭小屋门，打开X射线束。从此，喷油器必须从外壳远程操作。</li>
	<li>调整转速，直到生成稳定流。<ol>
<li>将转速 设置值 降低到 90。</li>
		<li>重复步骤 4.4.1，以 10 的增量降低 rpm 设置值 ，以减慢流速度，但仍保持稳定的流。对于硅胶润滑脂，使用了价值为 30 rpm 的油脂。 注：根据样本特征和X射线曝光时间，典型的转速范围在30-100 rpm（1=4 nL/s）之间变化。一般来说，rpm 应调整以产生最慢的流速（以尽量减少样本消耗），同时保持稳定的流。</li>
	</ol>
</li>
	<li>管理流动不畅的样本流。<ol>
<li>继续以 10 的增量增加 rpm 设置值 ，直到流变得更直。如果流不稳定，即使将 rpm 设置值 增加到 100 rpm 的最大值，也尝试通过实施下面描述的两种方法之一来提高稳定性。<ol>
<li>第一种方法：在样品流下方插入聚苯乙烯样品捕手，以帮助指导样品。此方法适用于带电的样本流。</li>
			<li>第二种方法：将吸气漏斗插入采样器。要向漏斗提供空气，将其连接到位于小屋中的空气出口管。此方法可以帮助指导和稳定样本流，而不受流的电荷限制。</li>
		</ol>
</li>
	</ol>
</li>
	<li>一旦实现了恒定稳定的流，开始数据收集，并根据正常的 X 射线晶体学实验优化探测器距离。 注：rpm 使用补充信息"Lipidico 设备计算器"中提供的转换表转换为流速。输入转速值、针直径和注射器体积，使用此计算器计算流速。</li>
</ol>

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MK适用于库塞尔设计。Kusel Design 是一家定制实验室设备开发商，由拉特罗贝大学的彼得·本特森博士和 ANSTO 的汤姆·卡拉多克-戴维斯博士共同参与，并开发了一种低成本设备，使澳大利亚同步加速器的 MX2 光束线能够进行高粘度研究。该装置是与卡拉多克-戴维斯博士密切协商后开发的。作者没有相互竞争的经济利益。

开发了一种替代的 HVI，非常适合在同步加速器源上进行 SX 实验。它比现有的高等健康高等疾病有两个关键优势。首先，在光束线上安装很容易，允许在传统晶体学和 SX 之间快速切换，在 MX2 上安装和对齐只需约 30 分钟。其次，用于种植晶体的样品注射器可直接用作注射液罐，从而限制样品转移过程中的浪费。已描述和演示更改样本的协议。与其他高射电相比，该设计无需复杂的气流来控制喷气?...

串行晶体学 （SX） 是一种最初在 X 射线自由电子激光器 （XFELs）1、2、3、4的背景下开发的技术。虽然固定目标方法可用于SX 5、6、7，但通常情况下，喷油器系统用于将晶体连续输送到X射线束。由于它结合了大量晶体的数据，SX避免了在实验过程中需要任何晶体对齐，并使数据能够在室温8，9时收集。在合适的喷油器的帮助下，晶体逐一流入X射线交互区，并在区域探测器9、10上收集由此产生的衍射数据。迄今为止，SX已经成功地解决了一些蛋白质结构1，11，12，13，包括晶体太小，无法测量使用传统的晶体学。它还利用XFEL的股秒脉冲持续时间，为时间解决的分子动力学提供了新的见解。通过使用光学激光源启动泵探针反应，对光系统II14、15、光活性黄蛋白16、17、细胞色素C氧化酶18以及细菌霍多普辛19、20、21进行了深入研究。这些研究已经探索了光激活后发生的电子转移动力学，证明了序列晶体学在理解时间解决的生物过程方面的巨大潜力。
连续晶体学的发展也越来越普遍同步加速器来源9，12，20，22，23，24。基于同步加速器的 SX 允许使用适当的喷油器系统在室温下有效地测量大量单个晶体。因此，这种方法适用于较小的晶体，因此，除了需要快速帧速探测器来收集数据外，还需要微聚焦光束。与传统的晶体学相比，SX 不涉及 X 射线束中单个晶体的安装和对齐。由于大量单个晶体的数据被合并，与传统晶体学相比，每个晶体接收的辐射剂量可以大大降低。同步加速器 SX 也可以应用于时间解决反应的研究，甚至到毫秒的机制，只要有足够高帧速率的探测器（例如，100 Hz 或更多）。在同步加速器上已经进行了几次连续晶体学实验，这些实验使用喷油器，这些喷油器最初是在XFEL源20、22、23号开发的。两种最常见的喷油器类型是气体动态虚拟喷嘴（GDVN）25和高粘性喷油器（HVI）9，24，26，27，28。GDVN 是注射低粘度液体样品的理想之选，但需要高流速才能实现稳定的流，这反过来又会导致高样品消耗率。相比之下，HVI 的适用于高粘度样品，从而以更低的流速生成稳定的流，从而降低样品消耗。因此，HVI喷油器倾向于在粘性载体更可取的情况下交付样品（例如，基于脂质的膜蛋白）和/或大量样品不可用。SX喷油器的使用通常具有挑战性，需要广泛的操作培训。它们还涉及冗长的样品传输协议，因为样品需要加载到专门的储层中，这通常与样品在"死体积"中丢失或连接中泄漏有关的风险很高。因此，在样品到达 X 射线束之前，优化喷油器设计以减轻任何损失是可取的。
最近，第一个SX结果使用利皮迪科23与酶靶点，使用Eiger 16M探测器发表。这种喷油器设计通过最大限度地减少从初始结晶到将晶体转移到喷油器中，然后将样品输送到 X 射线束中所涉及的步骤数量，从而限制样品浪费。本手稿描述并演示了从样品准备开始、进入注射过程，最后使用同一结晶容器收集数据的样品传输过程。还描述了喷油器的操作。

<table>
	<tbody>
		<tr>
			<td>Hen eggwhite lysozyme</td>
			<td>Sigma-Aldrich</td>
			<td>L6876</td>
			<td>Used to grow crystals for testing the injector and the crystals are transferred into silicon grease. https://www.sigmaaldrich.com/</td>
		</tr>
		<tr>
			<td>High vacuum silicon grease</td>
			<td>Dow Corning</td>
			<td>Z273554-1EA</td>
			<td>Used for testing of injector. https://www.sigmaaldrich.com/</td>
		</tr>
		<tr>
			<td>Injector needle (108 &#181;m ID)</td>
			<td>Hamilton</td>
			<td>part No: 7803-05</td>
			<td>www.hamiltoncompany.com</td>
		</tr>
		<tr>
			<td>Glass gas-tight syringes, 100 &#181;l</td>
			<td>Hamilton</td>
			<td>part no: 7656-01</td>
			<td>Syringes used for sample injection. www.hamiltoncompany.com</td>
		</tr>
		<tr>
			<td>LCP syringe coupler</td>
			<td>Formulatrix</td>
			<td>209526</td>
			<td>Syringe coupler to mix the samples</td>
		</tr>
		<tr>
			<td>Lipidico injector</td>
			<td>La Trobe Univerity/ANSTO</td>
			<td></td>
			<td>This is a specific piece of equipment that can be accessed through La Trobe University / ANSTO Australian Synchrotron Facility</td>
		</tr>
	</tbody>
</table>

lipidico injection protocol for serial crystallography measurements at the australian synchrotron

澳大利亚同步加速器公司已开发出一种用于进行连续晶体测量的设施。该设施采用了专用的高粘性喷油器Lipidico，作为大分子晶体学 （MX2） 光束线的一部分，可在室温下测量大量小晶体。该技术的目标是使晶体能够种植/转移到玻璃注射器中，直接用于喷油器的串行晶体成像数据收集。此喷油器的优点包括能够快速响应流速变化而不中断流。这种高粘度喷油器 （HVI） 存在几个限制，其中包括对允许的样品粘度限制为 +gt;10 Pa.s。流稳定性也可能是一个问题，具体取决于样本的特定属性。此处介绍了如何设置样品和操作喷油器以进行澳大利亚同步加速器的串行晶体学测量的详细协议。该方法演示了样品的准备，包括将酶晶体转移到高粘度介质（硅胶油脂）中，以及喷油器在 MX2 中用于数据收集的操作。

利皮迪科是作为替代交付系统在MX2（图1）上使用的HVI。它非常适合 SX，其中晶体要么生长在脂质立方相中，要么转移到高粘性惰性介质中。
为了证明喷油器应用硅胶油脂与溶酶晶体混合，用于在澳大利亚同步加速器的MX2光束线收集SX数据。要将喷油器安装在 MX2 光束线上，低温喷嘴将被移除，代之以 图 1中所示的喷油器。注射器样...

Watch this Scientific Journal Video about Lipidico Injection Protocol for Serial Crystallography Measurements at the Australian Synchrotron at JoVE.com

Lipidico Injection Protocol for Serial Crystallography Measurements at the Australian Synchrotron

该协议的目的是演示如何准备串行晶体学样本，以便于最近在澳大利亚同步加速器上委托的高粘度喷油器Lipidico上收集数据。

澳大利亚同步加速器串行晶体测量的利皮迪科注射协议

A facility for performing serial crystallography measurements has been developed at the Australian synchrotron. This facility incorporates a purpose built ...

lipidico-injection-protocol-for-serial-crystallography-measurements

Research

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Bioengineering

3.2K 次观看.  La Trobe University, Melbourne, Australia, 3086. 该协议的目的是演示如何准备串行晶体学样本，以便于最近在澳大利亚同步加速器上委托的高粘度喷油器Lipidico上收集数据。

视频: Lipidico Injection Protocol for Serial Crystallography Measurements at the Australian Synchrotron

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

创建的胶粘剂和可溶性梯度荧光显微镜成像细胞迁移

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

科研

生物工程

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

改进方法对血脑屏障的人体细胞为主，接触模型的制备

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III secretion system (T3SS) in Shigella flexneri minicells. It can be applicable to any project suitable for cryo-ET.

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

使用Tomoauto：一个协议，用于高通量自动化低温电子断层扫描

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

水凝胶的合成与防污性能作为膜的净水

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

Power Input Measurements in Stirred Bioreactors at Laboratory Scale

The power input in stirred bioreactors is an important scaling-up parameter and can be measured through the torque that acts on the impeller shaft during rotation. However, the experimental determination of the power input in small-scale vessels is still challenging due to relatively high friction losses inside typically used bushings, bearings and/or shaft seals and the accuracy of commercially available torque meters. Thus, only limited data for small-scale bioreactors, in particular single-use systems, is available in the literature, making comparisons among different single-use systems and their conventional counterparts difficult.
This manuscript provides a protocol on how to measure power inputs in benchtop scale bioreactors over a wide range of turbulence conditions, which can be described by the dimensionless Reynolds number (Re). The aforementioned friction losses are effectively reduced by the use of an air bearing. The procedure on how to set up, conduct and evaluate a torque-based power input measurement, with special focus on cell culture typical agitation conditions with low to moderate turbulence (100 &#60; Re &#60; 2&#183;104), is described in detail. The power input of several multi-use and single-use bioreactors is provided by the dimensionless power number (also called Newton number, P0), which is determined to be in the range of P0 &#8776; 0.3 and P0 &#8776; 4.5 for the maximum Reynolds numbers in the different bioreactors.

The power input in stirred bioreactors can be measured through the torque that acts on the impeller shaft during rotation. This manuscript describes how an air bearing can be used to effectively reduce friction losses observed in mechanical seals and improve the accuracy of power input measurements in small-scale vessels.

实验室规模搅拌生物反应器的功率输入测量

The power input in stirred bioreactors is an important scaling-up parameter and can be measured through the torque that acts on the impeller shaft during ...

Retroductal Nanoparticle Injection to the Murine Submandibular Gland

Two common goals of salivary gland therapeutics are prevention and cure of tissue dysfunction following either autoimmune or radiation injury. By locally delivering bioactive compounds to the salivary glands, greater tissue concentrations can be safely achieved versus systemic administration. Furthermore, off target tissue effects from extra-glandular accumulation of material can be dramatically reduced. In this regard, retroductal injection is a widely used method for investigating both salivary gland biology and pathophysiology. Retroductal administration of growth factors, primary cells, adenoviral vectors, and small molecule drugs has been shown to support gland function in the setting of injury. We have previously shown the efficacy of a retroductally injected nanoparticle-siRNA strategy to maintain gland function following irradiation. Here, a highly effective and reproducible method to administer nanomaterials to the murine submandibular gland through Wharton's duct is detailed (Figure 1). We describe accessing the oral cavity and outline the steps necessary to cannulate Wharton's duct, with further observations serving as quality checks throughout the procedure.

Local drug delivery to the submandibular glands is of interest in understanding salivary gland biology and for the development of novel therapeutics. We present an updated and detailed retroductal injection protocol, designed to improve delivery accuracy and experimental reproducibility. The application presented herein is the delivery of polymeric nanoparticles.

Retroductal 纳米微粒注射液对小鼠颌下腺的治疗

Two common goals of salivary gland therapeutics are prevention and cure of tissue dysfunction following either autoimmune or radiation injury. By locally ...

Automated Counterflow Centrifugal System for Small-Scale Cell Processing

Successful commercialization of gene and cell-based therapies requires manufacturing processes that are cost-effective and scalable. Buffer exchange and product concentration are essential components for most manufacturing processes. However, at the early stages of product development, these steps are often performed manually. Manual dead-end centrifugation for buffer exchange is labor-intensive, costly, and not scalable. A closed automated system can effectively eliminate this laborious step, but implementation can be challenging. Here, we describe a newly developed cell processing device that is suitable for small- to medium-scale cell processing and aims to bridge the gap between manual processing and large-scale automation. This protocol can be easily applied to various cell types and processes by modifying the flow rate and centrifugation speed. Our protocol demonstrated high cell recovery with shorter processing times in comparison to the manual process. Cells recovered from the automated process also maintained their proliferation rates. The device can be applied as a modular component in a closed manufacturing process to accommodate steps such as buffer exchange, cell formulation, and cryopreservation.

Automation is key to upscaling and cost management in cell manufacturing. This manuscript describes the use of a counterflow centrifugal cell processing device for automating the buffer exchange and cell concentration steps for small-scale bioprocessing.

用于小型细胞处理的自动逆流离心系统

Successful commercialization of gene and cell-based therapies requires manufacturing processes that are cost-effective and scalable. Buffer exchange and ...

Ceramic Omnidirectional Bioprinting in Cell-Laden Suspensions for the Generation of Bone Analogs

Structurally, bone tissue is an inorganic-organic composite containing metabolically active cells embedded within a hierarchical, highly mineralized matrix. This organization is challenging to replicate due to the heterogeneous environment of bone. Ceramic omnidirectional bioprinting in cell-suspensions (COBICS) is a microgel-based bioprinting technique that uniquely replicates the mineral and cellular structure of bone. COBICS prints complex, biologically relevant constructs without the need for sacrificial support materials or harsh postprocessing steps (e.g., radiation and high-temperature sintering), which are two of the biggest challenges in the additive manufacturing of bone mimetic constructs. This technique is enabled via the freeform extrusion of a novel calcium phosphate-based ink within a gelatin-based microgel suspension. The yield-stress properties of the suspension allow deposition and support the printed bone structure. UV crosslinking and nanoprecipitation then "lock" it in place. The ability to print nanostructured bone-mimetic ceramics within cell-laden biomaterials provides spatiotemporal control over macro- and micro-architecture and facilitates the real-time fabrication of complex bone constructs in clinical settings.

This protocol describes a 3D printing technique to fabricate bone-like structures by depositing a calcium phosphate ink in a gelatin-based granular support. Printed bone analogs are deposited in freeform, with flexibility for direct harvesting of the print or crosslinking within a living cell matrix for multiphasic constructs.

用于生成骨类似物的细胞悬浮液中的陶瓷全向生物打印

Structurally, bone tissue is an inorganic-organic composite containing metabolically active cells embedded within a hierarchical, highly mineralized ...

Experimental Quantification of Interactions Between Drug Delivery Systems and Cells In Vitro: A Guide for Preclinical Nanomedicine Evaluation

A major component of designing drug delivery systems concerns how to amplify or attenuate interactions with specific cell types. For instance, a chemotherapeutic might be functionalized with an antibody to enhance binding to cancer cells (&#34;targeting&#34;) or functionalized with polyethylene glycol to help evade immune cell recognition (&#34;stealth&#34;). Even at a cellular level, optimizing the binding and uptake of a drug carrier is a complex biological design problem. Thus, it is valuable to separate how strongly a new carrier interacts with a cell from the functional efficacy of a carrier&#39;s cargo once delivered to that cell.
To continue the chemotherapeutic example, &#34;how well it binds to a cancer cell&#34; is a separate problem from &#34;how well it kills a cancer cell&#34;. Quantitative in vitro assays for the latter are well established and usually rely on measuring viability. However, most published research on cell-carrier interactions is qualitative or semiquantitative. Generally, these measurements rely on fluorescent labeling of the carrier and, consequently, report interactions with cells in relative or arbitrary units. However, this work can be standardized and be made absolutely quantitative with a small number of characterization experiments. Such absolute quantification is valuable, as it facilitates rational, inter- and intra-class comparisons of various drug delivery systems-nanoparticles, microparticles, viruses, antibody-drug conjugates, engineered therapeutic cells, or extracellular vesicles.
Furthermore, quantification is a prerequisite for subsequent meta-analyses or in silico modeling approaches. In this article, video guides, as well as a decision tree for how to achieve in vitro quantification for carrier drug delivery systems, are presented, which take into account differences in carrier size and labeling modality. Additionally, further considerations for the quantitative assessment of advanced drug delivery systems are discussed. This is intended to serve as a valuable resource to improve rational evaluation and design for the next generation of medicine.

Experimental Quantification of Interactions Between Drug Delivery Systems and Cells In Vitro: A Guide for Preclinical Nanomedicine Evaluation

A workflow is demonstrated for the absolute quantification of drug carrier-cell interactions using flow cytometry to allow better rational evaluation of novel drug delivery systems. This workflow is applicable to drug carriers of any type.

体 外药物递送系统和细胞之间相互作用的实验定量：临床前纳米医学评估指南

A major component of designing drug delivery systems concerns how to amplify or attenuate interactions with specific cell types. For instance, a ...

一种减轻组织损伤的新型微创慢注射方法

A Novel Cell Injection Method with Minimum Invasion

Directly injecting cells into tissues is a necessary process in cell administration and/or replacement therapy. The cell injection requires a sufficient amount of suspension solution to allow the cells to enter the tissue. The volume of the suspension solution affects the tissue, and this can cause major invasive injury as a result of the cell injection. This paper reports on a novel cell injection method, called slow injection, that aims to avoid this injury. However, pushing out the cells from the needle tip requires a sufficiently high injection speed according to Newton's law of shear force. To solve the above contradiction, a non-Newtonian fluid, such as gelatin solution, was used as the cell suspension solution in this work. Gelatins solution have temperature sensitivity, as their form changes from gel to sol at approximately 20 &#176;C. Therefore, to maintain the cell suspension solution in the gel form, the syringe was kept cooled in this protocol; however, once the solution was injected into the body, the body temperature converted it to a sol. The interstitial tissue fluid flow can absorb excess solution. In this work, the slow injection technique allowed cardiomyocyte balls to enter the host myocardium and engraft without surrounding fibrosis. This study employed a slow injection method to inject purified and ball-formed neonatal rat cardiomyocytes into a remote area of myocardial infarction in the adult rat heart. At 2 months following the injection, the hearts of the transplanted groups showed significantly improved contractile function. Furthermore, histological analyses of the slow-injected hearts revealed seamless connections between the host and graft cardiomyocytes via intercalated disks containing gap junction connections. This method could contribute to next-generation cell therapies, particularly in cardiac regenerative medicine.

作者聚焦：一种侵入性最小的新型细胞注射方法  

This method eliminates any major invasion during cell injections caused by the cell suspension solution.

一种侵袭性最小的新型细胞注射方法

Directly injecting cells into tissues is a necessary process in cell administration and/or replacement therapy. The cell injection requires a sufficient ...