Name: automated counterflow centrifugal system for small-scale cell processing
Uploaded: 2019-12-12T12:00:00
Description: Watch this Scientific Journal Video about Automated Counterflow Centrifugal System for Small-Scale Cell Processing at JoVE.com

这项工作得到维多利亚州政府运营基础设施支助计划以及经济发展、就业、运输和资源部提供的维多利亚州政府技术券的支持。RL 是国家健康和医学研究委员会职业发展奖学金的获得者。AL 是澳大利亚研究生奖的获得者。

1. 制备试剂和细胞进行缓冲液交换
<ol><li>在 2 类层流罩中准备缓冲器（参见材料表）。使用注射器和针头组件，从 500 mL 盐袋中取出 50 mL 的盐水溶液。用50 mL的20%人血清白蛋白（HSA）代替，使2%的HSA在盐水中，这将作为洗涤缓冲液。</li>
	<li>从培养容器中取出细胞，并执行细胞计数，通过锥蓝色排除来确定起始细胞数量和生存能力。将细胞装入转包中。 注：在此协议中，Jurkat 细胞?...

<ol>
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描述的自动缓冲区交换协议简单且用户友好。但是，此协议中有几个关键步骤至关重要，需要特别注意。根据我们的经验，在处理较大的细胞（如 MSC（平均直径 10-15 μm）时，每次运行应至少包括 1 x 107个细胞，以实现最佳细胞恢复（图 4B）。处理较小的细胞，如Jurkat细胞（平均直径为±10μm），需要大约3 x 107个细胞来实现一个稳定的流化细胞床?...

现代医学的格局已经迅速改变，最近发展的基因和细胞为基础的疗法（GCT）。作为翻译研究领域发展最快的领域之一，GCT 行业也面临着独特且前所未有的挑战。除了强大的临床结果外，高效和具有成本效益的制造工艺对于GCT的商业成功至关重要，这在小规模制造1中尤其难以实现。当每批细胞只为一个病人生产几剂，而不是数百或数千次时，时间、劳动力和质量保证的成本被放大了。与异体细胞疗法不同，其制造过程更类似于抗体和重组蛋白的产生，自体细胞疗法通常作为小规模手术1生产。作为生物制药制造中较新的现象，目前小规模细胞加工的选择相当有限。
缓冲液交换对细胞制造至关重要。它是从培养培养体中去除细胞并浓缩用于冷冻保存或输液的下游过程之一。目前，小型细胞制造通常采用与学术研究环境类似的工艺，并依靠专门的洁净室来维持无菌3。手动下游工艺通常使用台式离心机对细胞进行颗粒和重悬浮，以减小体积和交换缓冲液。这些开放式工艺成本高昂（即人工和洁净室维护），制造能力有限，不适合商业生产2、3。
实施自动化已提出作为一种解决方案，以提高制造效率和实现商业规模生产2。在细胞产品中，不能通过用于生物制剂的传统方法（如伽马辐照或终端末端过滤）实现无菌。相反，部署了一个自动封闭系统，以减少污染的风险，操作人员依靠洁净室来维持无菌性。流程自动化还解决了可伸缩性问题，要么让多个系统并行运行（横向扩展），要么增加单个设备的处理能力（向上扩展），从而最大限度地减少操作员之间的可变性。此外，对自体疗法的成本建模分析表明，自动化可以降低制造成本5，6。然而，在一项自动干细胞临床试验中，没有发现任何成本效益，其中使用了7个自动化制造平台，这表明自动化的成本优势可能取决于单个制造过程。
自动化可以引入到现有的制造过程中，有不同的策略。这可以通过实现完全集成的平台或基于模块化的处理链来实现。有几个完全集成的平台可用于自体细胞制造，如CliniMACS神童（米尔滕尼生物泰克），可可（奥克坦生物技术）和量子（特鲁莫BCT）。这些集成平台通常被描述为"一盒 GMP"，对基础设施的要求较低，易于操作。但是，完全集成设置的制造能力可能会受到系统连接的孵化器的限制。例如，神童的培养能力仅限于其400mL室8，量子盒的极限表面积设置为2.1m2（相当于120 T175烧瓶）7，这可能不足以为需要更高细胞剂量9，10的患者。此外，神童和昆腾有一个共同的属性，限制他们的使用：操作单元被一批细胞在整个细胞扩展期间，从而限制了每个单元11可以制造的批次数。模块化自动化方法是创建一个具有多个模块化单元的制造链，以模拟商业制造工艺12、13。这种方法将培养装置与细胞清洗装置分离开来，从而最大限度地提高了制造效率。理想的加工设备是适应和可扩展的制造需求12。
反流离心（CFC）技术，可以追溯到20世纪70年代，在细胞处理方面有着悠久的历史。它通过平衡离心力和反流力来实现细胞浓度和分离。通常，细胞悬浮液在恒定流速下从细胞室的窄端进入，同时受到离心力的影响（图1A）。流体的流动与离心力方向相反。这称为逆流力，在细胞室内形成渐变。反流力则随着细胞室从锥形细胞室尖端变宽而减小。密度越高，直径越大的细胞具有较高的沉降率，因此它们达到向锥形细胞室尖端的力平衡。较小的颗粒可能达到向造型室底部的平衡，或太小，无法保留在腔室中，将被冲走。CFC技术主要以其在加工血液化产品中的应用而闻名，例如为树突状细胞疗法15、16分离单核细胞。在缓冲液交换方面，氟氯化碳技术只应用于大规模制造17，尚未用于小规模的自体细胞疗法制造。
为了满足小型细胞制造需要，最近开发了一种自动化的氟氯化碳装置（见材料表）。自动化细胞处理装置使用逆流离心技术去除细胞碎片，促进缓冲液交换。该设备使用一次性试剂盒进行缓冲交换，该试剂盒可无菌连接到细胞转移袋，从而允许在无菌封闭系统中处理细胞。在这里，我们研究在自动协议中使用逆流离心装置在哺乳动物细胞培养中执行缓冲交换。在这项研究中，我们分别测试了使用Jurkat细胞和美位位细胞（MSCs）的缓冲交换协议，分别对非附着细胞和附着细胞类型进行建模。Jurkat细胞是永生的T细胞，常用于研究急性T细胞白血病19、20。MSCs是成人干细胞，已在人类临床试验中研究过各种疾病9。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>20 ml Luer lock syringes</td>
			<td>BD</td>
			<td>302830</td>
		</tr>
		<tr>
			<td>20% Human serum albumin (HSA)</td>
			<td>CSL Behring</td>
			<td>AUST R 46283</td>
		</tr>
		<tr>
			<td>4-(Dimethylamino)benzaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>156477-25g</td>
		</tr>
		<tr>
			<td>500ml IV saline bag</td>
			<td>Fresenius Kabi</td>
			<td>K690521</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Thermo Fisher Scientific</td>
			<td>15240112</td>
		</tr>
		<tr>
			<td>Automated cell counter (Countess)</td>
			<td>Thermo Fisher Scientific</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Cell counting chamber slides</td>
			<td>Thermo Fisher Scientific</td>
			<td>C10228</td>
		</tr>
		<tr>
			<td>Cell stimulation cocktail (500x)</td>
			<td>Thermo Fisher Scientific</td>
			<td>00-4970-93</td>
		</tr>
		<tr>
			<td>Cell transfer bags</td>
			<td>Terumo</td>
			<td>T1BBT060CBB</td>
		</tr>
		<tr>
			<td>CellTiter AQueous One Solution Cell Proliferation Assay (MTS)</td>
			<td>Promega</td>
			<td>G3582</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
		</tr>
		<tr>
			<td>DMEM: F12 media</td>
			<td>Thermo Fisher Scientific</td>
			<td>11320082</td>
		</tr>
		<tr>
			<td>EnVision plate Reader</td>
			<td>Perkin Elmer</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10099141</td>
		</tr>
		<tr>
			<td>Human Interleukin 2 (IL2) Kit</td>
			<td>Perkin Elmer</td>
			<td>Al221C</td>
		</tr>
		<tr>
			<td>Luer (female) fittings</td>
			<td>CPC</td>
			<td>LF41</td>
		</tr>
		<tr>
			<td>PC laptop or PC tablet device</td>
			<td>ASUS</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Plate reader (SpectraMax i3)</td>
			<td>Molecular Device</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Recombinant Human IFN-&#947;</td>
			<td>PeproTech</td>
			<td>300-02</td>
		</tr>
		<tr>
			<td>Rotea counterflow centrifuge cell processing device</td>
			<td>Scinogy</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Rotea single-use processing kit</td>
			<td>Scinogy</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>RPMI media</td>
			<td>Thermo Fisher Scientific</td>
			<td>11875119</td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>ProSciTech</td>
			<td>420SS</td>
		</tr>
		<tr>
			<td>Trichloroacetic acide</td>
			<td>Sigma-Aldrich</td>
			<td>T6399-250g</td>
		</tr>
		<tr>
			<td>Trypan Blue stain</td>
			<td>Thermo Fisher Scientific</td>
			<td>T10282</td>
		</tr>
		<tr>
			<td>Trypsin digestion enzyme (TrypLE Express Enzyme)</td>
			<td>Thermo Fisher Scientific</td>
			<td>12604013</td>
		</tr>
	</tbody>
</table>

automated counterflow centrifugal system for small-scale cell processing

基因和基于细胞的疗法的成功商业化需要具有成本效益和可扩展性的制造过程。缓冲液交换和产品浓缩是大多数制造过程的基本组成部分。但是，在产品开发的早期阶段，这些步骤通常是手动执行的。用于缓冲区交换的手动死端离心是劳动密集型、成本高昂且不可扩展的。封闭的自动化系统可以有效地消除这一费力的步骤，但实施可能具有挑战性。在这里，我们描述了一种新开发的细胞处理设备，它适用于中小型细胞处理，旨在弥合手动处理与大规模自动化之间的差距。通过修改流速和离心速度，可轻松将该协议应用于各种细胞类型和过程。与手动工艺相比，我们的协议演示了高细胞恢复，处理时间更短。从自动化过程中恢复的细胞也保持其增殖率。该器件可作为模块化组件应用于封闭的制造工艺中，以适应缓冲液交换、细胞配方和冷冻保存等步骤。

在此协议中，我们使用 Jurkat 单元和 MSC 作为代表性示例来演示自动缓冲区交换过程。在此过程中，Jurkat 电池和 MSC 共享相同的处理步骤，在离心力和泵速度方面存在差异，以控制流速（表 1）。图 2显示了摄像机拍摄的具有代表性的图像，说明流化细胞床在缓冲液交换过程中如何出现。通常，流化的细胞床将类似于图2A中的图?...

Watch this Scientific Journal Video about Automated Counterflow Centrifugal System for Small-Scale Cell Processing at JoVE.com

Automated Counterflow Centrifugal System for Small-Scale Cell Processing

自动化是电池制造中提高规模和成本管理的关键。本手稿描述了使用逆流离心细胞处理装置实现缓冲液交换和细胞浓度步骤的自动化，用于小规模生物处理。

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 ...

该协议为小型电池制造提供了封闭式自动缓冲交换和浓缩解决方案。重要的是，缓冲交换能够以高恢复率和非常小的集中产品量执行。新用户可能难以执行所有自动化步骤。练习这些步骤编程和使用介质执行测试运行是熟悉设备的好方法。在开始缓冲液交换之前，在二类层流头中，使用注射器和针头组装从 500 毫升盐水袋中去除 50 毫升盐水溶液。用 50 毫升 20%的人血清白蛋白代替此体积，并收获其培养容器中感兴趣的细胞。计数后，使用注射器将细胞装入转移袋中，并使用 Luer 锁将转移袋连接到一次性处理袋。然后将先前准备好的洗涤缓冲袋连接到一次使用加工套件。要设置自动缓冲区交换，请打开设备的图形用户界面，然后单击"开始"按钮。单击"新"以创建新协议，然后单击控制以选择要打开和关闭的阀门、离心和泵速度、泵方向和操作触发器。设置所有参数后，单击"保存"。将连接好的适当行李挂在设备上，然后将一次使用加工套件放在机器上。然后单击连接按钮并下载已保存的程序。播放按钮亮起时，协议已准备好启动。要执行自动缓冲交换，请打开传输袋管的手动夹，然后单击"开始"以启动缓冲交换程序。在自动化步骤六结束时，由于现在空袋中空气触发气泡传感器，该过程将暂停。如果袋子确实是空的，请按下一步，将过程移动到下一步。自动化过程完成后，关闭所有管夹并打开设备的门。然后从设备中取出一次性套件，断开注射器，以便收集细胞以进行后续分析。通常，流化细胞床将在此图像中显示，细胞在圆锥体的中间和朝前累积，在腔室尖端有一个小空间，其中细胞不会累积，并在细胞加载入口内出现可见的开口。引入具有不同粘度或密度的新缓冲液时，可压缩流化细胞床。高流速可用于选择活细胞，因为死细胞更小、更轻，可以通过增加流速强制离开腔室。缓冲区交换处理过程的自动化过程比手动缓冲区交换处理过程短。在此分析中，手动和自动过程之间的恢复率对于两种测试的细胞类型都相似，并且细胞的生存能力不受这两种过程的影响。回收的细胞表现出类似的增殖率，产生细胞因子的能力，和伊迪奥活动，无论处理的类型。重要的是要记住，该协议仅作为一个指南，因此用户需要根据他们的细胞类型和缓冲区对流速和离心速度进行编程。缓冲交换是细胞制造中的中间步骤，可能跟随细胞培养扩化或低温保存和配方。目标是开发包围整个流程的方法。逆流离心是一个非常通用的平台。未来的研究将探讨细胞选择的应用，如死细胞去除和单核细胞分离从胆小球产品。

Research

JoVE Journal

Bioengineering

Postproduction Processing of Electrospun Fibres for Tissue Engineering

静电纤维组织工程的后期处理

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

科研

生物工程

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

一种多功能自动化平台微尺度细胞刺激实验

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

Gradient Strain Chip for Stimulating Cellular Behaviors in Cell-laden Hydrogel

在单元格拉丹水凝胶中刺激细胞行为的应变梯度芯片

Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single ...

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood

全自动离心微流体装置从全血超灵敏蛋白检测

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

模块化 DNA 设备的自动机器人液体处理组件

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications

设计和自动照明，培养和取样系统实现微生物光遗传学的应用

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular ...

Automated Slide Scanning and Segmentation in Fluorescently-labeled Tissues Using a Widefield High-content Analysis System

用 Widefield 高含量分析系统实现荧光标记组织的自动滑动扫描和分割

Automated slide scanning and segmentation of fluorescently-labeled tissues is the most efficient way to analyze whole slides or large tissue sections. ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

高通量自动 Microbioreactor 系统在 CHO 细胞模型 IgG1 生产中的应用

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System

Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System

利用自动高通量微流控系统对铜绿假单胞菌剪切应力的活细胞分析

A higher-throughput microfluidic in vitro bioreactor coupled with fluorescence microscopy has been used to study bacterial biofilm growth and morphology, ...

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

使用自动微生物反应器系统从中国仓鼠卵巢细胞中纯化和分析单克隆抗体

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...