新一代流式分选技术

niwanmao

以往的流式分选除了calibur采取机械阀，后来的仪器基本上采用电荷式分选，每秒可分选成千上万个细胞，纯度>90％，但对细胞活性影响较大，Owl公司发明了新一代流式分选技术，即采用微芯片技术制作了一个阀门，该阀门可以快速开关，使得无需电荷即可分选，使得细胞活力得到提高，而且较容易实现无菌分选。详细内容见下面的原文。也请@gcz5340 版主点评一下此文。
（转自http://www.genengnews.com/gen-articles/next-generation-flow-sorting/4904/）
＝＝＝＝＝＝原文＝＝＝＝＝
The purification of cellular populations and individual cells is pivotal for the reliable characterization of gene expression, many aspects of life science research, and the development of cellular therapies. Cell sorting routinely involves fluorescence-based separation through flow cytometry, which has proven superior to crude techniques such as differential sedimentation. Through this process, large numbers of cells are rapidly analyzed for specific fluorescence signatures. Traditionally, cell samples are parsed in charged aerosol droplets, which are electrostatically sorted, enabling purification at thousands of cells per second at purities often greater than 90%.
This technology enables exceptional specificity using multiple fluorescent signatures (e.g., cell surface labels, cell size, and granularity).

However, the contributions of current flow-sorting platforms are balanced against significant limitations, including: high processing pressures that can result in loss of function and/or cell death; sample processing speeds/volumes that make processing clinical-scale samples (>500 million cells) unfeasible; a high degree of technical expertise needed to manage device complexity; increased risk of sample contamination through the use of open systems; and user safety concerns when processing aerosolized patient samples.

These limitations, plus the high unit and sample processing costs, must be overcome to enable further clinical application and commercialization.
To address these issues, Owl biomedical developed a fully closed cell-sorting system. This microchip-based technology employs closed fluid path cartridges with aseptic ports that permit the straightforward administration and collection of cell samples. At the heart of the cartridge is a patented microchip capable of very high-frequency fluidic valving (Figure 1).

图1

Propelled by modest positive pressure, typically less than 0.2 atmospheres, cells pass through microchannels where laser-directed fluorescent signals are detected with photo-multiplier tubes. Upon identification of a positive target cell, the microchip valve opens, redirecting the cell to a collection chamber. Both positive and negative selected cells can then be retrieved from the cartridge and used for any number of downstream applications.

Improving Valve Speed

图2

Figure 2. Mechanism of microchip-based sorting: Labeled cell samples enter the chip from the input sample, as the cells approach the sort area each cell is analyzed. When a selected cell is identified a magnetic pulse opens and closes the valve and the cell is redirected to a collection chamber. An integrated single-crystal silicon spring returns the valve to its original position, and undesired cells are allowed to flow through.
One key design goal of all cell sorters is to maximize the speed at which the device can segment a stream of cells. In the case of Owl’s microchip-based technology, a fluidic valve determines the rate at which cells are isolated. Valve speed in a fluid microenvironment is in turn controlled by several factors, including the acceleration and magnitude of the opening and closing forces, and the inertia of the valve and the fluid surrounding it.

In the case of Owl technology, valve speed is controlled by its engineered magnetic properties and a powerful return-spring force, which serves to close the valve (Figure 2). Careful modeling and empirical testing has led to a design that allows μsec opening times, a user-selected sort collection delay, and μsec closing times.

A typical total cycle of around 50 μsec allows separation rates similar to a traditional droplet sorter, although with microchip-based sorting no aerosols or droplets are used.

Highly Viable Sorted Cells

Sorted cells are typically used for molecular analysis or sample preparation, for example, cellular expansion for research or therapeutic purposes. In such cases, those cells need to be in a healthy metabolic state ideally retaining their complete array of functional capabilities. Current flow cytometric sorting has distinct challenges in that respect due to technical requirements such as high pressure, extended shear rate, severe decompression upon aerosolization, and impact trauma during cell collection. Often, these factors result in cell isolates with compromised function and/or viability.

Using Owl’s microchip-based technology, the pressure applied to cells is minimal and the trauma associated with droplet sorting is removed, resulting in high cell viability. In addition, a wide variety of cell types have been sorted using the Owl technology, all with a high retention of cell functionality. For example, antigen-primed T cells have been shown to retain their cancer-specific cytotoxic capabilities in chromium-release assays.

Microchip-Based Sorting

图3

Figure 3. Sorting target cells from dilute whole blood or PBMCs: Pre-sort fraction of diluted whole blood with CD4+ cells (A) Post-sort fraction showing enrichment of CD-4 positive cells (B) Pre-sort fraction of the spiked MART-1-specific T-cell clone into PBMCs (C) Post-sort fraction showing enrichment of MART-1 positive antigen specific T cells (D).


While the utility of microchip-based cell separation is being tested in many different applications, current studies show its utility in processing clinical samples for diagnostic and therapeutic applications. For these purposes it is best to process the samples with as little manipulation as possible.

Here, CD4+ cells were sorted from a diluted whole blood preparation by adding a fluorescent CD4 antibody marker to 8 mL PBS and 2 mL of whole blood. Results show that while the presorted fraction contained less than 0.01% CD4 positive cells, the sorted fraction contained more than 91% CD4 positive cells (Figures 3A and B). The effective purification was close to 10,000-fold in a single-step process and with a simple no-lyse, no-wash sample preparation.

Investigations have also been done to study the ability of antigen-specific T cells from a patient’s own blood to recognize tumor cells and trigger an immune system response. To explore the feasibility of this clinically important strategy, cells from a MART-1-specific T cell clone were spiked into a patient’s peripheral blood mononuclear cells (PBMCs), stained with a PE-conjugated tetramer loaded with a MART-1 peptide, and then sorted using the Owl’s microchip-based sorting technology.

Results show tetramer-positive cells were enriched from less than 1% to greater than 95% (Figures 3C and D). Importantly, sorted cells maintained their ability to kill MART-1+ tumor cells and to proliferate in vitro.

Conclusions

Acceleration of research for effective cancer and cellular therapies has increased the need for cell-sorting technologies that are safe, efficient, and easy to use. Owl’s cartridge-based closed system has effectively demonstrated its capability to sort a wide variety of cells at high speeds without impacting cell viability. This platform offers the additional advantage of protecting sample integrity while permitting quick sample-to-sample changeover—a feature that avoids the inter-sample cleaning and validation required for traditional cell sorting.

That this technology does not utilize sheath fluids opens the future potential for sequential sorting of large numbers of samples within a short timeframe, making practical clinical applications a possibility. In addition, combining the underlying microchip technology with other cell isolation techniques such as magnetic beads has the potential to empower processing of samples greater than 1 billion cells.

gcz5340

    倪老师的搜索能力很强啊，这是美国owl公司研发的一款分选仪器，现在已被德国美天旎公司收购，明年会正式上市。

    微芯片控制的高速机械式分选，我第一次看到时也眼前一亮，相对于电荷式分选我觉得他有以下优势：
1、得到高活性细胞
    充电对细胞损伤还是蛮大的，特别一些免疫细胞可能会活化，原代细胞可能活性很低，机械式分选能解决这一难题。

2、生物安全性好
    电荷式分选产生的液滴形成气溶胶，飘出来可能产生生物安全性问题。所以流式分选操作人员风险还是蛮大的，特别是一些做病毒或者医院病人样本的分选，而国内对这一块的重视还不够。owl是全封闭式的，不用产生液滴，这是一个很大的优势。

3、卡槽式设计避免管道污染或样本间交叉污染
    Owl的液流系统全部是在图中的卡槽中，卡槽有很多种，临床用户可能选择一次性的，科研用户考虑成本可能选择可重复使用的卡槽。分选的时候就是把样本放在卡槽里，插入机器中就可以分选。传统分选仪万一机器管道污染了就很麻烦，各种冲洗，听说Influx是可以更换管路的，但应该成本高、操作也比较麻烦；传统流式分选不同样本时，两管之间要冲洗很长时间，但有时候还是很难避免交叉污染的情况。卡槽式设计能解决这个问题。

    以上是亮点，个人觉得可能的缺陷有：
1、看上面介绍好像暂时只能单路分选，一次只能分选一群细胞；
2、卡槽式设计是亮点，虽然有的卡槽可以重复使用，但是耗材的成本可能是用户比较关心的；
3、文中提到无鞘液设计，不知道单细胞流怎么形成的，效果如何。

    个人觉得概念确实很新很有创意，实用性如何还需经市场考验。

tanweifeng

赞一个 分析的很到位！学习了

dragonhzqsmmu

gcz5340 发表于 2013-8-25 10:27

                               
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倪老师的搜索能力很强啊，这是美国owl公司研发的一款分选仪器，现在已被德国美天旎公司收购，明年会正 ...

无鞘液流流式细胞之前好像millipore的Guava流式细胞就是采用的这个技术，但那个是分析型流式细胞仪，优点是对环境影响小，因为长时间跑下来没有什么废液，具体的机制好像是通过微量负压泵抽吸（毛细管作用）可能会导致管路拥堵，但据说也好清洗，反冲加超声毛细管路就OK，但个人觉得这个技术要用于分析低丰度或者极低丰度的目标细胞可能不是很合适，因为这种情况往往原始标本的体积就很大，芯片由于容纳废液有限做这种低丰度细胞的分选可能会比较吃力

另外单路分选将来也可能再扩展成多路，或者通过对芯片进行设计成多路闸门，这个在微流控里面已经不是什么难题了，或者把激光分选的点设在单路分选后，形成树枝扩展结构？

feireiro

好像SONY公司也有一款基于芯片的分选产品，不过国内貌似没有上市。看来以后分选的市场越来越热闹了，除了BD和BC这两家之外，Bio-Rad也有基于空气激发的分选仪，加上美天旎，SONY, Fludigim，JSAN等，大家以后可以讨论的话题更多了。

dragonhzqsmmu

feireiro 发表于 2013-8-26 09:14

                               
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好像SONY公司也有一款基于芯片的分选产品，不过国内貌似没有上市。看来以后分选的市场越来越热闹了，除了BD ...

sony的那款是不是这个链接里面的，http://www.sony.com.cn/news_cent ... ology/1955_3968.htm

基于蓝光技术开发的

http://finance.people.com.cn/n/2013/0110/c348883-20157065.html

报价据说是2000万日元

feireiro

是的，型号是SH8000，听说台湾有卖，不过价格很贵，好像也要将近30万美金。