作者要感谢Nilou Sarah Arden和Zhong Zhao对这份手稿的建设性评论。作者还要感谢Lindsey Brown在这个项目期间提供的重要投入。这项工作的部分内部资金和支持由CDER关键路径计划（CA #1-13）和CDER制造科学，卓越创新中心计划（Berilla-CoE-19-49）提供。该项目部分得到了美国食品和药物管理局生物技术产品办公室的实习/研究参与计划的支持，该计划由橡树岭科学与教育研究所通过美国能源部和FDA之间的机构间协议进行管理。
本出版物反映了作者的观点，不应被解释为代表FDA的观点或政策。

1. AWS 的准备工作
注:该协议是使用一个声束电泳室开发的。但是，实验室规模的 AWS 有 5 个浊度探头，如果需要，可以串联操作 4 个声束室。
<ol><li>将浊度电缆连接到标记为 F、1、2、3、4 的相应端口（例如，图 1c中的浊度探头 1 和图 2a中的端口 1），并将腔室电源 BNC 电缆连接到标记为 1、2、3、4 的 AWS 系统背面（图 2b）。 注:在步骤2.6中充满液体之前，请勿将腔室电源BNC电缆的另一端（图3c）连接到声束室的背面（图3d）。如果腔室电源打开时腔室中没有液体，则会损坏腔室中的压电传感器，不再起作用。</li>
	<li>将浊度探头插入浊度计和温度计外壳中，然后拧紧螺钉（图1c和图3）。</li>
	<li>通过进料泵将进料管连接到进料浊度端口的输入端（图4a）（图1b至1c）。</li>
	<li>将Y型管从进料浊度端口的输出端（图4b）连接到声束室的入口端口（图4c）。</li>
	<li>通过stage1泵将Stage1管从声束室的废物端口（图4d）连接到细胞收集容器（图1d通过1e到1f）。</li>
	<li>将管道从声电泳室的渗透口（图4e）连接到探头1浊度端口的输入端（图4f）。</li>
	<li>将收获管从探头1浊度端口（图4g）连接到产品收集容器（图1c至1g）。</li>
</ol>2. 使用HCCF为系统预热
注意:该方案是使用在化学定义的培养基（ActiCHO P与6mM L-谷氨酰胺）中培养的CHO-K1细胞开发的，产生IgG1型单克隆抗体VRC0113， 可能需要针对其他细胞系和产物进行调整。该方案中使用的HCCF是在7-8天的CHO-K1培养物结束时从振荡瓶或生物反应器过程中获得的。
<ol><li>通过打开 AWS 背面和前面的电源开关来打开 AWS。</li>
	<li>打开计算机并双击相关软件的桌面图标（请参见 材料表）。在"读数"面板中，按"开始测试"按钮启动数据记录（图5）。启动数据记录后，收集的所有数据都将记录在可导出的电子表格中，直到测试停止。</li>
	<li>将进料管端连接到正在搅拌的HCCF容器中。</li>
	<li>通过在"进料泵图像"的"控制"屏幕上输入泵速并按键盘上的"Enter"来启动进水泵。确保在进水泵图像右侧的灰色框中正确选择了"泵方向箭头图标"（顺时针或逆时针），以将HCCF从容器泵入声电泳室。点击进水泵图像下方灰色框中"打开"字样的"三角形图标"，以"启动"水泵（图6a）。 注:最大泵速为 10 L/h （167 mL/min），但建议在此步骤中使用标称流速 60 mL/min，以快速填充管道和腔室，而不会在开始分离之前使腔室溢出。</li>
	<li>在声束室填充过程中，通过观察"百分比还原板"（图5c）来监测进料浊度测量值。如果在HCCF容器中充分混合HCCF，则在腔室装载期间浊度值将保持一致。</li>
	<li>一旦液体位于声吸室背面的压电换能器上方，在进料泵图像下方的灰色框中按"关闭"键以停止泵。将BNC电源线（图3c）连接到声束室（图3d）。 注:每个声束孔的滞留量约为190 mL。</li>
</ol>3. AWS 的运营
<ol><li>一旦声束槽充满并且BNC电源线连接到声束室的背面，将进料泵的速率更改为所需的运行速率（图6a 和 表1）。应测试多个进料泵速率，以确定给定过程的理想操作参数。</li>
	<li>通过将电源模块中的压杆滑动至10 W（图6d），然后按Stage 1框右侧的"打开"图标（图6c），打开stage1压电电源。根据制造商的建议，使用 10 W，这是 CHO 电池的推荐功率设置。这将产生2 MHz的固定工作频率。几秒钟后，细胞将开始在声束室中的波节点处明显聚集（图7a）。</li>
	<li>一旦细胞开始沉降到声束室的底部（图7b），根据细胞密度和进料泵速率以适当的速率启动stage1泵（图6b）。根据制造商的优化和稳态操作电子表格，可以使用以下等式根据填充的细胞质量和进料流量计算第一级泵速率 <img alt="Equation 1" src="/files/ftp_upload/61161/61161equ01.jpg"><ol>
<li>要计算填充的细胞质量，请用空的15 mL管（或其他与离心兼容的管）去垢。用进料填充管子，并记录进料管的总重量。将试管在3，700 x g下离心10分钟。将上清液倒入单独的容器中。用细胞沉淀测量管的重量。填充的细胞质量百分比的进料材料=（卧螺离心管重量/填充管重量）×100%。</li>
	</ol>
</li>
	<li>当声束室溢出进入浊度探头1时，监测阶段1浊度的浊度曲线（图8）。</li>
	<li>随着细胞分离的继续，细胞去除效率 <img alt="Equation 2" src="/files/ftp_upload/61161/61161equ02.jpg"> 将提高。</li>
	<li>此外，监测进料和阶段1之间的温差，因为随着细胞分离的继续，温差会增加（图9）。当使用较高细胞密度的进料时，温度可能会升高，但通常可以通过改变进料流速来降低。 注:当使用多个声束电泳槽时，可以通过浊度探头将管子从以前的声束槽依次连接到当前声束槽的输入端。此外，还可以通过级泵将声束槽底部的载物台管连接到细胞收集容器。总共可以串联四个声束槽，以便在必要时获得最佳的细胞去除效率。</li>
</ol>4. 终止 AWS
<ol><li>运行结束后，通过分别按进料泵下方的灰色框内的 "关闭" 和"第 1 阶段泵图像"来停止进水泵和阶段 1 泵，以停止进水泵和级 1 级泵。</li>
	<li>通过按 Stage 1 盒右侧的 "关闭" 并断开 BNC 电源线，关闭腔室电源。</li>
	<li>取收集到的产品收获材料进行进一步澄清和纯化程序，如深度过滤和色谱方法。丢弃细胞收获材料。</li>
	<li>通过将废气管放入空容器中，将管子与声吸管室入口和渗透口断开，并从泵头释放废气管，从而排出声液槽中的剩余流体。</li>
	<li>重新连接管道，将废气管放回泵头，并使用 60 mL/min 的进料泵速率和 60 mL/min 的阶段泵速率从进料管末端流经 DI 水。继续 15–20 分钟。丢弃流经。</li>
	<li>使用进料泵将70%异丙醇（IPA）泵入管道和腔室15-20分钟。丢弃流经。</li>
	<li>用去离子水重复清洁程序，通过使用进料泵15-20分钟来清除管道和腔室。丢弃流经。</li>
	<li>从浊度探头和声束室中拆卸管道，并用70%的IPA清洁该区域。</li>
	<li>拆卸浊度探头，并用70%IPA清洁浊度探头内部。让所有部件风干，然后重新组装以备下次使用。 注:声束腔室为一次性使用而制造，但如果按照本协议所述正确处理和清洁，可以重复使用。</li>
</ol>

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作者在本手稿中描述的产品中没有经济利益，也没有其他要披露的内容。

描述是一个分步协议，用于实施实验室规模的AWS，以初步澄清来自CHO细胞系HCCF的模型单克隆抗体。如代表性结果所示，在初级澄清中使用 AWS 可有效进行单元澄清和产品回收。此外，低水平的维护和操作要求以及放大生产能力允许在初级澄清中具有更广泛的应用潜力。
重要的是，具有代表性的结果表明，进料泵速率对于细胞的分离至关重要。此外，由于在浊度测量中检测高细胞密度的限制，工作细胞密度是使用 AWS 系统时要考虑的另一个因素。由于在运行高细胞密度进料时浊度探针会饱和，因此最好通过离线测量进料和澄清的细胞培养液的细胞密度来计算细胞分离效率。通过精确的离线澄清测量，在解决这些问题时可以考虑的一种过程策略是串联使用多个腔室。虽然该协议主要侧重于使用单个腔室，但该系统可以操作三个额外的腔室，以便对细胞进行顺序澄清，从而最大限度地减少对细胞状况的影响并导致高产物回收率。此外，其他参数（例如 AWS 的功率和单元去除流速）可以针对特定的单元类型或操作模式进一步优化。总体而言，建议在实施 AWS 之前使用这些注意事项优化操作参数和策略。
在众多应用潜力中，AWS在连续生物处理中的应用前景广阔。因为AWS可以取代离心并大幅减少过滤器表面积14，使用AWS将能够为后续过滤和色谱过程提供恒定的无细胞材料流，与连续生物制造兼容。由于这种兼容性以及灌注技术对高细胞密度（例如，>50 x 106 个细胞/mL）和更长的培养持续时间（例如，>14 天）的可用性，AWS 除了初级澄清之外，还有可能开发出一种新的连续细胞出血策略。
在某些情况下，在稳态操作期间产生的高达30%的蛋白质在细胞出血材料15，16中被除去。此外，要将去除的单克隆抗体与标准收获的材料混合在一起，必须满足某些质量属性。当不符合这些规格时，结果可能是材料被拒收，从而影响产品产量。为了补偿此类产品损失，可以在长期灌注过程中以稳态条件对使用 AWS 的细胞出血材料进行连续澄清17。这种策略可以减少出血材料中的产物损失，并利用更多的蛋白质产生。此外，如果需要更高的细胞密度和生产率，使用AWS进行连续澄清后的细胞可能会被重新添加到生物反应器中。因此，使用 AWS 持续澄清细胞渗漏材料可能会提供在给定过程中提高产量和/或减少二次过滤器表面积的机会。
总而言之，AWS 的实用性不仅限于简单的初级澄清，还可能适用于连续生物工艺中的应用程序，从而提高制造率和运营灵活性。

在涉及分泌的治疗蛋白的生物制造过程中，一个关键步骤是从收获的细胞培养液（HCCF）中去除生物质。传统上，生物制造商在生产单克隆抗体时采用离心和深度过滤作为其主要澄清方法1。然而，离心可能导致细胞上的高剪切应力，导致HCCF中的细胞碎片增加。这可能导致过滤器在过滤过程中结垢，并导致额外的污染物后过滤，从而降低下游色谱效率1，2，3。此外，为特定过程定制离心机可能成本高昂，并且可能需要额外的连接到原位清洗和原位灭菌系统，这也可能是可扩展性的限制因素。使用深度过滤器可以补偿离心的限制，并利用一次性技术4。然而，深度过滤器主要用作二次澄清，因为它们无法承受高细胞培养密度5。或者，已采用切向流动过滤（TFF）细胞保留装置来减轻剪切应力，但可能会遇到诸如膜极化和收获率低等挑战6。使用离心加深度过滤或TFF引起的上述问题为改进HCCF的初级澄清过程创造了机会。
声学分离被引入为一种可用于从具有高质量蛋白质产物的细胞培养物中收获分泌蛋白质的技术7，8。声学分离是通过与悬浮流体和保留粒子相互作用的多维驻波的传播和反射来实现的9，10。这些粒子经历三种力:流体阻力，重力和声辐射。当每个力彼此均匀相对，达到平衡时，粒子被悬浮并捕获在超声波驻波10内。在细胞培养悬浮液中，细胞保持在驻波的这个压力节点平面内，节点随着细胞的聚结而增长，最终这些细胞节点簇从引力9中落下。然后将这些沉淀的细胞从培养基中取出，从而允许将澄清的培养基泵出以进行进一步的下游处理。超声波作为分离方法的利用已开始转化为生物学应用，从脂质颗粒和红细胞11的分离到哺乳动物灌注细胞培养12。凭借其通过避免离心、深度过滤或TFF来降低成本、劳动力和细胞应激的相对能力，生物制造商正在探索使用声学分离的潜在应用。
本研究为操作台式声波分离器（AWS）以澄清CHO细胞培养提供了通用方案，提供了代表性数据，并演示了如何实现有效的细胞澄清和产物回收。

<table>
	<tbody>
		<tr>
			<td>Acoustophorectic chamber</td>
			<td>Pall</td>
			<td>CAS-AC-K1 Cadence acoustic chamber kit</td>
			<td></td>
		</tr>
		<tr>
			<td>ActiCHO P</td>
			<td>GE</td>
			<td>SH31025.01</td>
			<td>powder medium</td>
		</tr>
		<tr>
			<td>AWS</td>
			<td>Pall</td>
			<td>CAS-SYS (60500101-SP)</td>
			<td></td>
		</tr>
		<tr>
			<td>Cadence Acoustic Separator Software</td>
			<td>Pall</td>
			<td>Cadence Acoustic Separator Interface Ver. 1.0.4</td>
			<td></td>
		</tr>
		<tr>
			<td>CHO-K1 cells</td>
			<td>VRC</td>
			<td>VRC01</td>
			<td></td>
		</tr>
		<tr>
			<td>Computer</td>
			<td>Dell</td>
			<td>Latitude 3470</td>
			<td>Windows 7, 64 bit OS</td>
		</tr>
		<tr>
			<td>Isopropanol (70%)</td>
			<td>LabChem</td>
			<td>LC157605</td>
			<td>20L prepped 70% IPA</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Corning</td>
			<td>25-005-CV</td>
			<td>200 mM stock solution</td>
		</tr>
		<tr>
			<td>Masterflex L/S 14 tubing</td>
			<td>Cole-Parmer</td>
			<td>96400-14</td>
			<td>peroxide-cured silicone tubing, 25ft</td>
		</tr>
		<tr>
			<td>Masterflex L/S 16 tubing</td>
			<td>Cole-Parmer</td>
			<td>96400-16</td>
			<td>peroxide-cured silicone tubing, 25ft</td>
		</tr>
		<tr>
			<td>Tubing set</td>
			<td>Pall</td>
			<td>CAS-FP-K1 Tubing set</td>
			<td></td>
		</tr>
		<tr>
			<td>Turbidity probe</td>
			<td>Pall</td>
			<td>CAS-TS-S1 (60500106)</td>
			<td></td>
		</tr>
	</tbody>
</table>

primary clarification of cho harvested cell culture fluid using an acoustic separator

初级澄清是生物制造过程中必不可少的步骤，用于从收获的细胞培养液中的治疗产物中初始去除细胞。虽然离心或过滤等传统方法被广泛用于细胞去除，但这些过程的设备占地面积大，操作可能涉及污染风险和过滤器结垢。此外，传统方法可能不适合用于初级澄清的连续生物工艺方案。因此，研究了使用声（声）波的替代应用，以连续地将细胞与细胞培养液分离。本研究提出了一个详细的方案，用于使用实验室规模的声波分离器（AWS）从CHO细胞生物反应器收获中对含有单克隆IgG1抗体的培养液进行一次分离。AWS 提供了具有代表性的数据，并演示了如何实现有效的单元澄清和产品恢复。最后，讨论了 AWS 在连续生物处理中的潜在应用。总体而言，本研究为在 CHO 细胞培养的初级澄清中实施 AWS 提供了实用的通用方案，并进一步描述了其在连续生物工艺中的应用潜力。

如协议中所述，AWS用于以12.4 x 106 个细胞/ mL的密度澄清HCCF，如图 1所示。将进料泵设置为3.5 mL /分钟（5 L /天），表示细胞出血速率在10-20 L培养物的假定合适范围内。当HCCF进入AWS室时，来自进料浊度探头的浊度测量值保持一致，约为1，000-1，100 NTU，而来自探头1的浊度探头的测量值保持在40-50 NTU左右（图8）。使用两种测量方法，去除细胞效率
<img alt="Equation 2" src="/files/ftp_upload/61161/61161equ02.jpg">
计算并平均为95%。结果发现，40–50 NTU 的浊度测量是可以达到的最低浊度水平，因此无法从额外的 AWS 室中进一步分离。
虽然AWS可以在较低的流速下高效分离，但将HCCF在腔室内保持较长时间会导致温度升高，这在选择较低流速时应该是一个考虑因素。 图9 是HCCF在进入声束室之前和声学分离后以3.5 mL / min的进料流速进行的温差示例，显示由于声室内的时间延长，温度升高了>6°C。
运行高细胞密度收获（即>20 x 106 个细胞/ mL）时的另一个重要考虑因素是浊度探针的饱和度。进料浊度探头的浊度测量值在4，400 NTU以上饱和（图10），这可能导致在计算细胞去除效率时被低估。
为了测试不同进料速率对细胞澄清的影响，在不同进料速率下测量了细胞去除效率。如图 11所示，随着进料泵速率的提高，细胞去除效率从~100%显著下降到57%。一般来说，进料泵速度越慢，细胞澄清效果越好。但是，建议对每种应用优化进给速率。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61161/61161fig01.jpg"> 图 1:AWS 设置。一旦使用软件（a）打开泵，HCCF就通过进料泵（b）通过进料浊度探头（c）进入，然后进入AWS室（d）。在腔室内，声力将细胞从波的节点中捕获并导致聚集。浮力降低导致细胞通过重力下降，细胞通过stage1泵（e）从声束室的废物端口被移除到细胞收获瓶（f），而澄清的材料通过腔室的渗透口离开探针1浊度探针（c）到产品收获瓶（g）。<a href="https://www.jove.com/files/ftp_upload/61161/61161fig01large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61161/61161fig02.jpg"> 图 2:AWS 系统的背面。浊度探头和腔室通过浊度探头以太网（a）和腔室电源 BNC（b）电缆连接到 AWS 系统背面的相应端口。此外，计算机通过PC以太网电缆（c）连接。 <a href="https://www.jove.com/files/ftp_upload/61161/61161fig02large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61161/61161fig03.jpg"> 图 3:浊度探头和外壳。每个浊度探头（a）必须正确插入相应的浊度计和温度计外壳（b）并用螺钉拧紧。腔室功率BNC电缆（c）只有在腔室中的压电换能器充满流体后，才应连接到声束室（d）的背面。探头指示如下:F = 进料浊度，1 = 探头1浊度，2、3 和 4 是未使用的探头（或可用于序列化过程）。 <a href="https://www.jove.com/files/ftp_upload/61161/61161fig03large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61161/61161fig04.jpg"> 图4:浊度外壳和声学室之间的连接。进料管通过进料泵连接到进料浊度端口（a）的输入端。进料浊度口（b）的输出通过Y型管连接到声束室（c）的入口口。Stage1管通过stage1泵从声束室（d）的废物口连接到细胞收集容器。声束室（e）的渗透端口连接到探头1浊度端口（f）的输入。收获管从探头1浊度端口（g）外部连接到产品收集容器。 <a href="https://www.jove.com/files/ftp_upload/61161/61161fig04large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61161/61161fig05.jpg"> 图 5:声学分离器软件中的读数面板。该程序有两个面板，"读数"和"控件"。在"读数"面板中，浊度（a）、温度（b）和百分比降低（c）被监测。要启动数据记录，需要单击"开始测试"按钮（d），这会将按钮的颜色更改为绿色。 <a href="https://www.jove.com/files/ftp_upload/61161/61161fig05large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61161/61161fig06.jpg"> 图6:程序中的控制面板。在"控制"面板中，泵可以打开或关闭，并且泵送速率可以改变进料（a）和其他阶段（b）。此外，压电传感器上的腔室电源可以打开或关闭（c），并用滑动杆（d）进行更改。实验使用10 W，因为它是制造商推荐的CHO电池的推荐功率设置。 <a href="https://www.jove.com/files/ftp_upload/61161/61161fig06large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61161/61161fig07.jpg"> 图 7:AWS 商会。一旦细胞进入腔室，声力就会将细胞困在波的节点中，并导致细胞聚集在（a）中。这些细胞簇的大小增加，直到它们失去浮力并最终通过引力（b）稳定下来。接下来，通过stage1泵将沉降的细胞从声束电泳室的废物端口中取出到细胞收获瓶（c）中。产品通过渗透口（d）离开腔室，而HCCF通过入口口（e）连续填充腔室。 <a href="https://www.jove.com/files/ftp_upload/61161/61161fig07large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/61161/61161fig08.jpg"> 图 8:进料泵速率为 3.5 mL/min 的 12 x 106 个细胞/mL CHO 细胞培养物的浊度测量。进料浊度（蓝色）约为1，000 NTU，从阶段1浊度计（橙色）渗出的渗透物为40-60 NTU。 <a href="https://www.jove.com/files/ftp_upload/61161/61161fig08large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/61161/61161fig09.jpg"> 图 9:进料泵速率为 3.5 mL/min 的 12 x 106 个细胞/mL CHO 细胞培养物的温度测量值。进料温度（蓝色）约为 21 °C，从第 1 阶段浊度计（橙色）流出的渗透物约为 27 <a href="https://www.jove.com/files/ftp_upload/61161/61161fig09large.jpg" target="_blank">°C。请单击此处查看此图的放大版本。</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/61161/61161fig10.jpg"> 图 10:高细胞密度样品的浊度测量示例。当细胞密度>20×106 个细胞/mL时，进料浊度测量在4，400 NTU的最大值处饱和，导致细胞去除效率被低估。 <a href="https://www.jove.com/files/ftp_upload/61161/61161fig10large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/61161/61161fig11.jpg"> 图 11:电池去除效率比较。随着进料速率的增加，细胞分离降低。因此，随着进料速率的增加，细胞澄清效率降低。 <a href="https://www.jove.com/files/ftp_upload/61161/61161fig11large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>参数</td>
			<td>规范</td>
		</tr>
<tr>
<td>流量</td>
			<td>0 – 10 升/小时</td>
		</tr>
<tr>
<td>压力范围</td>
			<td>0 – 2 巴 （0 – 30 磅/平方英寸）</td>
		</tr>
<tr>
<td>进料流体温度</td>
			<td>0 – 40 °C （32 – 104 °F）</td>
		</tr>
<tr>
<td>工作温度</td>
			<td>0 – 40 °C （32 – 104 °F）</td>
		</tr>
</tbody></table>表1:操作条件。 AWS 制造商针对流速、压力范围、进料流体和工作温度的建议操作条件。

Watch this Scientific Journal Video about Primary Clarification of CHO Harvested Cell Culture Fluid using an Acoustic Separator at JoVE.com

Primary Clarification of CHO Harvested Cell Culture Fluid using an Acoustic Separator

这里介绍的是使用声学分离器对CHO细胞培养进行初步澄清的方案。该协议可用于摇瓶培养物或生物反应器收获的初级澄清，并且具有在灌注生物反应器操作期间连续澄清细胞出血材料的潜在应用。

使用声学分离器对CHO收获的细胞培养液进行初步澄清

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested ...

primary-clarification-cho-harvested-cell-culture-fluid-using-an

Research

JoVE Journal

Bioengineering

5.0K 次观看.  U.S. Food and Drug Administration. 这里介绍的是使用声学分离器对CHO细胞培养进行初步澄清的方案。该协议可用于摇瓶培养物或生物反应器收获的初级澄清，并且具有在灌注生物反应器操作期间连续澄清细胞出血材料的潜在应用。

视频: Primary Clarification of CHO Harvested Cell Culture Fluid using an Acoustic Separator

Developing Custom Chinese Hamster Ovary-host Cell Protein Assays using Acoustic Membrane Microparticle Technology

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to the complexity of development on alternate platforms. BioScale's proprietary Acoustic Membrane MicroParticle (AMMP) technology allows sandwich immunoassays to be easily developed for use on the ViBE platform, providing better sensitivity, reproducibility, and automated operation. Provided as an example, this protocol outlines the procedure for developing a custom Chinese Hamster Ovary- Host Cell Protein (CHO-HCP) assay. The general principles outlined here can be followed for the development of a wide variety of immunoassays.
An AMMP assay measures antigen concentration by measuring changes in oscillation frequency caused by the binding of microparticles to the sensor surface to calculate. It consists of four major components: (1) a cartridge that contains a functionalized eight sensor chip (2) antibody labeled magnetic microparticles, (3) hapten tagged antibody that binds to the surface of the functionalized chip (4) samples containing the antigen of interest. BioScale's biosensor is a resonant device that contains eight individual membranes with separate fluidic paths. The membranes change oscillation frequency in response to mass accumulating on the surface and this frequency change is used to quantitate the amount of added mass.
To facilitate use in a wide variety of immunoassays the sensor is functionalized with an anti-hapten antibody. Assay specific antibodies are modified through the covalent conjugation of a hapten tag to one antibody and biotin to the other. The biotin label is used to bind the antibody to streptavidin coupled magnetic beads which, in combination with the hapten-tagged antibody, are used to capture the analyte in a sandwich. The complex binds to the chip through the anti-hapten/hapten interaction. At the end of each assay run the sensors are cleaned with a dilute acid enabling the sequential analysis of columns from a 96-well plate.
Here, we present the method for developing a custom CHO-HCP AMMP assay for bioprocess development. Developing AMMP assays or modifying existing assays into AMMP assays can provide better performance (reproducibility, sensitivity) in complex samples and reduced operator time. The protocol shows the steps for development and the discussion section reviews representative results. For a more in-depth explanation of assay optimization and customization parameters contact BioScale. This kit offers generic bioprocess development assays such as Residual Protein A, Product titer, and CHO-HCP.

Development of custom assays on the ViBE platform for more sensitive, reproducible, automated results in complex matrices is described. The universal cartridge allows assays to be easily adapted for use with custom assays. This versatility enables rapid development and validation of novel assays or automated versions of existing manual assays, exemplified in this video.

开发自定义的中国仓鼠卵巢宿主细胞蛋白检测使用声膜微粒子技术

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to ...

科研

生物工程

Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming

Correlating gene expression with cell behavior is ideally done at the single-cell level. However, this is not easily achieved because the small amount of labile mRNA present in a single cell (1-5% of 1-50pg total RNA, or 0.01-2.5pg mRNA, per cell 1) mostly degrades before it can be reverse transcribed into a stable cDNA copy. For example, using standard laboratory reagents and hardware, only a small number of genes can be qualitatively assessed per cell 2. One way to increase the efficiency of standard laboratory reverse transcriptase (RT) reactions (i.e. standard reagents in microliter volumes) comprising single-cell amounts of mRNA would be to more rapidly mix the reagents so the mRNA can be converted to cDNA before it degrades. However this is not trivial because at microliter scales liquid flow is laminar, i.e. currently available methods of mixing (i.e. shaking, vortexing and trituration) fail to produce sufficient chaotic motion to effectively mix reagents. To solve this problem, micro-scale mixing techniques have to be used 3,4. A number of microfluidic-based mixing technologies have been developed which successfully increase RT reaction yields 5-8. However, microfluidics technologies require specialized hardware that is relatively expensive and not yet widely available. A cheaper, more convenient solution is desirable. The main objective of this study is to demonstrate how application of a novel "micromixing" technique to standard laboratory RT reactions comprising single-cell quantities of mRNA significantly increases their cDNA yields. We find cDNA yields increase by approximately 10-100-fold, which enables: (1) greater numbers of genes to be analyzed per cell; (2) more quantitative analysis of gene expression; and (3) better detection of low-abundance genes in single cells. The micromixing is based on acoustic microstreaming 9-12, a phenomenon where sound waves propagating around a small obstacle create a mean flow near the obstacle. We have developed an acoustic microstreaming-based device ("micromixer") with a key simplification; acoustic microstreaming can be achieved at audio frequencies by ensuring the system has a liquid-air interface with a small radius of curvature 13. The meniscus of a microliter volume of solution in a tube provides an appropriately small radius of curvature. The use of audio frequencies means that the hardware can be inexpensive and versatile 13, and nucleic acids and other biochemical reagents are not damaged like they can be with standard laboratory sonicators.

We describe a novel method for increasing cDNA yield from single-cell quantities of mRNA in otherwise standard laboratory reverse transcription reactions. The novelty resides in the use of a micromixer, which utilizes the phenomenon of acoustic microstreaming, to mix fluids at microliter scales more effectively than shaking, vortexing or trituration.

增加从单细胞的数量标准实验室逆转录反应的mRNA的cDNA产量，使用声Microstreaming

Correlating gene expression with cell behavior is ideally done at the single-cell level.  However, this is not easily achieved because the small amount of ...

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

三维细胞培养模式的测量肿瘤细胞浸润间质流体流动的影响

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

利用双水相系统的细胞共培养图案

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

电池挤压作为一种强大的，微流控细胞内传送平台

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion.

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, in vitro.

微流体流钱伯斯再造使用血止血建模和血小板输注<em&gt;体外</em

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

倒胶体晶体聚（乙二醇）脚手架制作:肝脏组织工程三维细胞培养平台

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

用集成电极直接量化 Transendothelial 电阻的 Organ-on-chip 系统的制作与验证

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

Handling and Assessment of Human Primary Prostate Organoid Culture

This paper describes a detailed protocol for three-dimensional (3D) culturing, handling, and evaluation of human primary prostate organoids. The process involves seeding of epithelial cells sparsely in a 3D matrix gel on a 96-well microplate with media changes to cultivate expansion into organoids. Morphology is then assessed by whole-well capturing of z-stack images. Compression of z-stacks creates a single in-focus image from which organoids are measured to quantify a variety of outputs, including circularity, roundness, and area.DNA, RNA, and protein can be collected from organoids recovered from the matrix gel. Cell populations of interest can be assessed by organoid dissociation and flow cytometry. Formalin-fixation-paraffin-embedding (FFPE) followed by sectioning is used for the histological assessment and antibody staining. Whole-mount immunofluorescent staining preserves organoid morphology and facilitates observation of protein localization in organoids in situ. Commercial assays that are traditionally used for 2D monolayer cells can be modified for 3D organoids. Used together, the techniques in this protocol provide a robust toolbox to quantify prostate organoid growth, morphologic characteristics, and expression of differentiation markers.

Here, we present a protocol to guide human primary prostate organoid handling then suggest endpoints to assess phenotype. Seeding, culture maintenance, recovery from matrix gel, morphologic quantification, embedding and sectioning, FFPE sectioning, whole-mount staining, and application of commercial assays are described.

人原发性前列腺组织培养的处理与评价

This paper describes a detailed protocol for three-dimensional (3D) culturing, handling, and evaluation of human primary prostate organoids. The process ...

Screening and Identification of Small Peptides Targeting Fibroblast Growth Factor Receptor2 using a Phage Display Peptide Library

The human Fibroblast Growth Factor Receptor (FGFR) family comprises four members, namely, FGFR1, FGFR2, FGFR3, and FGFR4, that are involved in various biological activities including cell proliferation, survival, migration and differentiation. Several aberrations in the FGFR signaling pathway, owing to mutations or gene amplification events, have been identified in various types of cancers. Hence, recent research has focused on developing strategies involving therapeutic targeting of FGFRs. Current FGFR inhibitors at various stages of pre-clinical and clinical development include either small molecule inhibitors of tyrosine kinases or monoclonal antibodies, with only a few peptide- based inhibitors in the pipeline. Here, we provide a protocol using phage display technology to screen small peptides as antagonists of FGFR2. Briefly, a library of phage-displayed peptides was incubated in a plate coated with FGFR2. Subsequently, unbound phage was washed off by TBST (TBS + 0.1% [v/v] Tween-20), and bound phage was eluted with 0.2 M glycine-HCl buffer (pH 2.2). The eluted phage was further amplified and used as input for the next round of biopanning. Following three rounds of biopanning, the peptide sequences of individual phage clones were identified by DNA sequencing. Finally, the screened peptides were synthesized and analyzed for affinity and biological activity.

Herein, we present a detailed protocol for screening small peptides that bind to FGFR2 using a phage display peptide library. We further analyze the affinity of the selected peptides toward FGFR2 in vitro and its ability to suppress cell proliferation.

使用噬菌体显示肽库筛选和识别针对纤维细胞生长因子受体2的小肽

The human Fibroblast Growth Factor Receptor (FGFR) family comprises four members, namely, FGFR1, FGFR2, FGFR3, and FGFR4, that are involved in various ...