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Bio-Design and Manufacturing  2022 Vol.5 No.2 P.358-370

http://doi.org/10.1007/s42242-021-00167-y


3D spiral channels combined with flexible micro-sieve for high-throughput rare tumor cell enrichment and assay from clinical pleural effusion samples


Author(s):  Jie Cheng, Lina Zhang, Yiran Zhang, Yifei Ye, Wenjie Zhao, Lingqian Zhang, Yuang Li, Yang Liu, Wenchang Zhang, Hongyan Guo, Mingxiao Li, Yang Zhao & Chengjun Huang

Affiliation(s):  Institute of Microelectronics, Chinese Academy of Sciences, Beijing 100029, China ; more

Corresponding email(s):   zhaoyang@ime.ac.cn, huangchengjun@ime.ac.cn

Key Words:  Cell enrichment, High throughput, Pleural effusion, Liquid biopsy, 3D printing


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Jie Cheng, Lina Zhang, Yiran Zhang, Yifei Ye, Wenjie Zhao, Lingqian Zhang, Yuang Li, Yang Liu, Wenchang Zhang, Hongyan Guo, Mingxiao Li, Yang Zhao & Chengjun Huang . 3D spiral channels combined with flexible micro-sieve for high-throughput rare tumor cell enrichment and assay from clinical pleural effusion samples[J]. Journal of Zhejiang University Science D, 2022, 5(2): 358-370.

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author="Jie Cheng, Lina Zhang, Yiran Zhang, Yifei Ye, Wenjie Zhao, Lingqian Zhang, Yuang Li, Yang Liu, Wenchang Zhang, Hongyan Guo, Mingxiao Li, Yang Zhao & Chengjun Huang ",
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publisher="Zhejiang University Press & Springer",
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A1 - Wenjie Zhao
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A1 - Yang Liu
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Abstract: 
The sieving and enrichment of rare tumor cells from large-volume pleural effusion (PE) samples is a promising technique for cell-based lung tumor diagnosis and drug tests, which features high throughput and recovery, purification, as well as viability rates of rare target cells as the prerequisites for high sensitivity, specificity, and accuracy of tumor cell analysis. In this paper, we propose a three-dimensional (3D) sieving method for rare tumor cell enrichment, which effectively eliminates the "dead zones" in traditional two-dimensional (2D) cell filters with a dimension-raising strategy to satisfy the requirements mentioned above. The prototype device was combined with a funnel-shaped holder, a flexible micropore membrane in the middle, and a 3D spiral fluid channel covered on the membrane as a three-layer ice-creaming cone composite structure. Driven by gravity alone, the device performed as follows: (1) 20-fold throughput compared with the 2D commercial plane cell filter, which was up to 20 mL/min for a threefold dilution of whole blood sample; (2) high recovery rates of 84.5% ± 21%, 86% ± 25%, 83% ± 14% for 100, 1000, and 10 000 cells/mL, respectively, in 30 mL phosphate buffer saline (PBS) sample, and a 100% positive detection rate in the case of ≤ 5 A549 cells in 1 mL PBS; (3) a typical purification rate of 85.5% ± 9.1%; and (4) a viability rate of > 93%. In the demonstration application, this device effectively enriched rare target cells from large volumes (> 25 mL) of clinical pleural effusions. The following results indicated that tumor cells were easy-to-discover in the enriched PE samples, and the proliferation capability of purified cells was (> 4.6 times) significantly stronger than that of unprocessed cells in the subsequent 6-day culture. The above evaluation indicates that the proposed easily reproducible method for the effective execution of rare cell enrichments and assays is expected to become a practical technique for clinical cell-based tumor diagnosis.

中科院微电子所黄成军等 | 像手冲咖啡一样容易:3D螺旋通道集成的细胞筛,用于临床胸腔积液样本中稀有肿瘤细胞的高通量富集与分析

本研究论文聚焦3D螺旋通道集成的细胞筛用于临床胸腔积液样本中稀有肿瘤细胞的高通量富集与分析。肺癌病人的胸腔积液中潜藏的稀有肿瘤细胞可能携带着丰富的肿瘤信息。通过对于这些信息的获取和分析,有望被用于指导肺癌临床诊断和制定药物治疗方案。然而,由于这些细胞的稀有性,从100 mL量级的胸腔积液中高通量、高回收率地富集出高纯度、高活性的疑似肿瘤细胞依然存在较大挑战。现有的基于肿瘤细胞尺寸的细胞筛分法简单有效,但是在过滤过程中孔之间存在“流体死区空间”,存在易堵塞、细胞滤不干净等不足。


论文作者受日常手工冲调咖啡的启发,提出一种将传统2D过滤,变成3D过滤的升维策略,较好地克服了上述不足。基于该思路,设计了一种漏斗型的3D螺旋通道集成的细胞筛器件(简称“3D细胞筛”),并用于从胸腔积液中对稀有肿瘤细胞的富集筛选。


该3D细胞筛装置是由漏斗形支架、中间的柔性微孔膜以及扣压在膜上的3D螺旋型微流体通道相结合构成的三层锥型复合结构,如图1所示。它的特色之处在于利用膜上的3D螺旋通道引导含有稀有细胞的待分析液体样本在膜上螺旋流动,从而在流动中不断将小细胞(背景细胞)和液体滤出,而大细胞(目标细胞)最终流向锥尖被最终回收,达到高纯度富集目的。


实验结果发现,只需简单将待分析样品倒入该3D细胞筛,不需要离心或者压力驱动,即可达到如下性能指标:(1) 通量方面:对于全血样品三倍稀释的过滤通量最高可达20 mL/min,比2D方法提高了20倍;(2) 回收率方面:30 mL PBS中100、1000、10000 cells/mL的回收率可分别达到(84.5±21)%、(86±25)%、(83±14)%,同时,1 mL PBS仅有5个A549细胞的样品中阳性检出率均能达到100%;(3)纯度方面:典型达到(85.5±9.1)%;(4)细胞活性:大于93%。


进一步的,在该3D细胞筛的临床样本实验中,实现了从25 mL临床胸腔积液中快速高效富集稀有肿瘤细胞的能力。而且在富集后细胞样品的随后6天培养过程中,发现富集纯化后细胞的增殖能力超过未富集的细胞约4.6倍。


通过本研究,作者开发了一套简单高效的稀有细胞富集筛选方法,并在全血样品和临床胸腔积液样品中进行初步实验验证。本研究成果未来有望发展成为一种辅助临床肿瘤诊断和精准医学的实用化工具。


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