Name: primary clarification of cho harvested cell culture fluid using an acoustic separator
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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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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

Universidad San Sebastian

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

Preparation of Primary Acute Lymphoblastic Leukemia Cells in Different Cell Cycle Phases by Centrifugal Elutriation

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum deprivation; chemicals which arrest cells at different cell cycle phases; or the use of mitotic shake-off which exploits their reduced adherence. However, all of these have disadvantages. For example, serum starvation works well for normal cells but less well for tumor cells with compromised cell cycle checkpoints due to oncogene activation or tumor suppressor loss. Similarly, chemically-treated cell populations can harbor drug-induced damage and show stress-related alterations. A technique which circumvents these problems is counterflow centrifugal elutriation (CCE), where cells are subjected to two opposing forces, centrifugal force and fluid velocity, which results in the separation of cells on the basis of size and density. Since cells advancing through the cycle typically enlarge, CCE can be used to separate cells into different cell cycle phases. Here we apply this technique to primary acute lymphoblastic leukemia cells. Under optimal conditions, an essentially pure population of cells in G1 phase and a highly enriched population of cells in G2/M phases can be obtained in excellent yield. These cell populations are ideally suited for studying cell cycle-dependent mechanisms of action of anticancer drugs and for other applications. We also show how modifications to the standard procedure can result in suboptimal performance and discuss the limitations of the technique. The detailed methodology presented should facilitate application and exploration of the technique to other types of cells.

This protocol describes the use of centrifugal elutriation to separate primary acute lymphoblastic leukemia cells into different cell cycle phases.

离心淘洗制备不同细胞周期的原发性急性淋巴细胞白血病细胞

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum ...

科研

癌症研究

Isolation and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular infiltrate and epithelial cell activation. The progenitor cell population during such inflammatory reactions has mostly been investigated using single surface markers, either by histological analysis or by flow cytometry-based techniques. However, novel surface markers identified various functionally distinct subsets within the liver progenitor/stem cell compartment. The method presented here describes the isolation and detailed flow cytometry analysis of progenitor subsets using novel surface marker combinations. Moreover, it demonstrates how the various progenitor cell subsets can be isolated with high purity using automated magnetic and FACS sorting-based methods. Importantly, novel and simplified enzymatic dissociation of the liver allows for the isolation of these rare cell populations with a high viability that is superior in comparison to other existing methods. This is especially relevant for further studying progenitor cells in vitro or for isolating high-quality RNA to analyze the gene expression profile.

Liver injuries are accompanied by progenitor cell expansion that represents a heterogeneous cell population. Novel classification of this cellular compartment allows for the distinguishing of multiple subsets. The method described here illustrates the flow cytometry analysis and high purity isolation of various subsets that can be used for further assays.

分离和新型表面标记组合来标识肝祖亚群富集

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular ...

发育生物学

A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. However, to achieve robust sample manipulation it is necessary to address device integration with the macroscale environment. To realize repeatable, sensitive particle separation with microfluidic devices, this protocol presents a complete automated and integrated microfluidic platform that enables precise processing of 0.15&#8211;1.5 ml samples using microfluidic devices. Important aspects of this system include modular device layout and robust fixtures resulting in reliable and flexible world to chip connections, and fully-automated fluid handling which accomplishes closed-loop sample collection, system cleaning and priming steps to ensure repeatable operation. Different microfluidic devices can be used interchangeably with this architecture. Here we incorporate an acoustofluidic device, detail its characterization, performance optimization, and demonstrate its use for size-separation of biological samples. By using real-time feedback during separation experiments, sample collection is optimized to conserve and concentrate sample. Although requiring the integration of multiple pieces of equipment, advantages of this architecture include the ability to process unknown samples with no additional system optimization, ease of device replacement, and precise, robust sample processing.

This protocol describes a system architecture for performing automated small volume (0.15&#8211;1.5 ml) particle separations using a microfluidic device, and discusses methods to optimize acoustofluidic device performance and operation.

微流体平台精密小批量来样加工及它的使用尺寸分离生物颗粒的声微型

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. ...

生物工程

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

高通量单细胞的分离和文化的微流体平台

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic properties, enable bio imaging and control cell phenotype. A critical yet inadequately addressed issue is the significant number of particles that remain unbound after cell labeling which cannot be readily removed by conventional centrifugation. This leads to an increase in bio imaging background noise and can impart transformative effects onto neighboring non-target cells. In this protocol, we present an inertial microfluidics-based buffer exchange strategy termed as Dean Flow Fractionation (DFF) to efficiently separate labeled cells from free NPs in a high throughput manner. The developed spiral microdevice facilitates continuous collection (&#62;90% cell recovery) of purified cells (THP-1 and MSCs) suspended in new buffer solution, while achieving &#62;95% depletion of unbound fluorescent dye or dye-loaded NPs (silica or PLGA). This single-step, size-based cell purification strategy enables high cell processing throughput (106 cells/min) and is highly useful for large-volume cell purification of micro/nanoparticle engineered cells to achieve interference-free clinical application.

This protocol describes the use of an inertial microfluidics-based buffer exchange strategy to purify micro/nanoparticle engineered cells with efficient depletion of unbound particles.

微流控缓冲液更换为无干扰微/纳米颗粒细胞工程

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic ...

Microfluidic Acoustophoresis for Flowthrough Separation of Gram-Negative Bacteria using Aptamer Affinity Beads

This article describes the fabrication and operation of microfluidic acoustophoretic chips using a microfluidic acoustophoresis technique and aptamer-modified microbeads that can be used for the fast, efficient isolation of Gram-negative bacteria from a medium. This method enhances the separation efficiency using a mix of long, square microchannels. In this system, the sample and buffer are injected into the inlet port through a flow controller. For bead centering and sample separation, AC power is applied to the piezoelectric transducer via a function generator with a power amplifier to generate acoustic radiation force in the microchannel. There is a bifurcated channel at both the inlet and outlet, enabling simultaneous separation, purification, and concentration. The device has a recovery rate of &gt;98% and purity of 97.8% up to a 10x dose concentration. This study has demonstrated a recovery rate and purity higher than the existing methods for separating bacteria, suggesting that the device can separate bacteria efficiently.

This paper describesthe fabrication and operation of microfluidic acoustophoretic chips using the microfluidic acoustophoresis technique and aptamer-modified microbeads that can be used for fast, efficient isolation of Gram-negative bacteria from a medium.

微流体声导术，使用适配子亲和磁珠对革兰氏阴性菌进行流式分离

This article describes the fabrication and operation of microfluidic acoustophoretic chips using a microfluidic acoustophoresis technique and ...

工程学

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae zygotes result from the fusion of haploid cells of distinct mating type (MATa, MATalpha) and give rise to corresponding stable diploids that successively generate as many as 20 diploid progeny as a result of their strikingly asymmetric mitotic divisions 2. Zygote formation is orchestrated by a complex sequence of events: In this process, soluble mating factors bind to cognate receptors, triggering receptor-mediated signaling cascades that facilitate interruption of the cell cycle and culminate in cell-cell fusion. Zygotes may be considered a model for progenitor or stem cell function.
Although much has been learned about the formation of zygotes and although zygotes have been used to investigate cell-molecular questions of general significance, almost all studies have made use of mating mixtures in which zygotes are intermixed with a majority population of haploid cells 3-8. Many aspects of the biochemistry of zygote formation and the continuing life of the zygote therefore remain uninvestigated.
Reports of purification of yeast zygotes describe protocols based on their sedimentation properties 9; however, this sedimentation-based procedure did not yield nearly 90% purity in our hands. Moreover, it has the disadvantage that cells are exposed to hypertonic sorbitol. We therefore have developed a versatile purification procedure. For this purpose, pairs of haploid cells expressing red or green fluorescent proteins were co-incubated to allow zygote formation, harvested at various times, and the resulting zygotes were purified using a flow cytometry-based sorting protocol. This technique provides a convenient visual assessment of purity and maturation. The average purity of the fraction is approximately 90%. According to the timing of harvest, zygotes of varying degrees of maturity can be recovered. The purified samples provide a convenient point of departure for "-omic" studies, for recovery of initial progeny, and for systematic investigation of this progenitor cell.

Flow Cytometry-based Purification of S. cerevisiae Zygotes

To purify zygotes of S. cerevisiae, haploid cells of opposite mating type were engineered to express red or green fluorescent proteins, co-incubated to allow zygote formation, and fractionated using a flow cytometry-based protocol. The highly-enriched fraction enables subsequent "-omic" studies, recovery of initial progeny, and systematic investigation of zygote morphogenesis.

流式细胞仪分离纯化<em&gt; S.酵母</em&gt;合子

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

生物学

Size Exclusion Chromatography for Separating Extracellular Vesicles from Conditioned Cell Culture Media

Extracellular vesicles (EVs) are nano-sized lipid-membrane bound structures that are released from all cells, are present in all biofluids, and contain proteins, nucleic acids, and lipids that are reflective of the parent cell from which they are derived. Proper separation of EVs from other components in a sample allows for characterization of their associated cargo and lends insight into their potential as intercellular communicators and non-invasive biomarkers for numerous diseases. In the current study, oligodendrocyte derived EVs were isolated from cell culture media using a combination of state-of-the-art techniques, including ultrafiltration and size exclusion chromatography (SEC) to separate EVs from other extracellular proteins and protein complexes. Using commercially available SEC columns, EVs were separated from extracellular proteins released from human oligodendroglioma cells under both control and endoplasmic reticulum (ER) stress conditions. The canonical EV markers CD9, CD63, and CD81 were observed in fractions 1-4, but not in fractions 5-8. GM130, a protein of the Golgi apparatus, and calnexin, an integral protein of the ER, were used as negative EV markers, and were not observed in any fraction. Further, when pooling and concentrating fractions 1-4 as the EV fraction, and fractions 5-8 as the protein fraction, expression of CD63, CD81, and CD9 in the EV fraction was observed. The expression of GM130 or calnexin was not observed in either of the fraction types. The pooled fractions from both control and ER stress conditions were visualized with transmission electron microscopy and vesicles were observed in the EV fractions, but not in the protein fractions. Particles in the EV and protein fractions from both conditions were also quantified with nanoparticle tracking analysis. Together, these data demonstrate that SEC is an effective method for separating EVs from conditioned cell culture media.

The protocol here demonstrates that extracellular vesicles can be adequately separated from conditioned cell culture media using size exclusion chromatography.

用于从条件细胞培养基中分离细胞外囊泡的体积排阻色谱

Extracellular vesicles (EVs) are nano-sized lipid-membrane bound structures that are released from all cells, are present in all biofluids, and contain ...

神经科学

Harvesting and Disaggregation: An Overlooked Step in Biofilm Methods Research

Biofilm methods consist of four distinct steps: growing the biofilm in a relevant model, treating the mature biofilm, harvesting the biofilm from the surface and disaggregating the clumps, and analyzing the sample. Of the four steps, harvesting and disaggregation are the least studied but nonetheless critical when considering the potential for test bias. This article demonstrates commonly used harvesting and disaggregation techniques for biofilm grown on three different surfaces. The three biofilm harvesting and disaggregation techniques, gleaned from an extensive literature review, include vortexing and sonication, scraping and homogenization, and scraping, vortexing and sonication. Two surface types are considered: hard non-porous (polycarbonate and borosilicate glass) and porous (silicone). Additionally, we provide recommendations for the minimum information that should be included when reporting the harvesting technique followed and an accompanying method to check for bias.

This paper details methods that demonstrate three common biofilm harvesting and disaggregation techniques on two surface types, ruggedness testing of a harvesting method and minimum information to consider when choosing and optimizing harvesting and disaggregation techniques to increase reproducibility.

收获和分解:生物膜方法研究中被忽视的步骤

Biofilm methods consist of four distinct steps: growing the biofilm in a relevant model, treating the mature biofilm, harvesting the biofilm from the ...

Leukemic Subpopulation Harvest: A Method for Spatial Separation of Leukemic Cell Subpopulations from 2D Co-culture

Source: Slone, W. L., et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/t/53645">Modeling Chemotherapy Resistant Leukemia In Vitro.</a> J. Vis. Exp. (2016).
This video describes a method for spatial separation of three leukemic cell subpopulations: the suspended leukemic cells freely floating in the media, phase bright leukemic cells adhered to the surface of the mesenchymal stromal cell monolayer, and the phase dim leukemic cells that have migrated beneath the mesenchymal stromal cell monolayer from 2D co-culture.

白血病亚群收获:一种从 2D 共培养中空间分离白血病细胞亚群的方法

Source: Slone, W. L., et al. Modeling Chemotherapy Resistant Leukemia In Vitro. J. Vis. Exp. (2016).
This video describes a method for spatial separation ...