Discovery of the correlation between the suspended membrane capacitance and adherent morphology of single cells enriching from clinical pleural effusion revealed by a microfluidic impedance flow cytometry

https://doi.org/10.1016/j.snb.2022.132487Get rights and content

Highlights

  • Systematically 3D sieved, proliferated single cells from PEs, and electrical characterized.

  • Er quantified for cells' adherent elongation ratio.

  • Our high-throughput impedance flow cytometry characterized ~65,400 primary single cells' suspended membrane capacitance.

  • The correlation between cells' Er and membrane capacitance was revealed and involved adherent information in suspension.

Abstract

Cellular adherent morphology and suspended electrical property are two important intrinsic biophysical features of single cells in two states. However, few studies reported their relationship due to lacking systematic methods. Here, we proposed a toolchain for enriching, proliferating single cells from pleural effusions (PEs), and characterizing their adherent morphologies and suspended inherent electrical properties. Our 3D cell sieving device was employed to enrich rare tumor cells from every 50 mL clinical PEs. After proliferated, ten samples were enrolled, whose cells' adherent morphologies were quantified with the elongation ratio (Er). Our microfluidic impedance flow cytometry was developed to characterize ~65,400 suspended single cells' electrical properties (e.g., Csm). Subsequently, we experimentally found that the Csm of 5 spindle-like (mainly Er> 2) samples were all quantified as focused above 1.5 μF/cm2, whereas others' were all focused around 1–1.5 μF/cm2 for 5 round-like (mainly Er≤ 2) samples. Spearman rhos were introduced to further quantify this potential correlation from aspects of proportions (Csm> 1.5 μF/cm2, Er> 2), average, and median, noting as 0.758 (p = 0.011), 0.760 (p = 0.011), and 0.744 (p = 0.014), respectively. Those results revealed a significant correlation between single cells' Er and Csm─which means that the underlying correlation between cells' two label-free biophysical properties presented in two states was discovered.

Introduction

As an explicit biological feature, cell morphologies provide detailed information on cell types, physiological/pathological cellular conditions [1], or even disease statuses [2], [3], [4]. Likewise, electrical properties (e.g., specific membrane capacitance Csm and cytoplasm conductivity σcyto), as intrinsic cellular properties, have emerged as a critical label-free biophysical indicator [5], [6] for cell states assessment [7], [8] and classification [9], [10]. These two features have been extensively applied for cell biology study and clinical diagnostics from two different dimensions.

However, is there any relationship between cells' morphologies and electrical properties? Various studies have been taken to find out pieces of the answer, and some "direct correlation" for the same cell states was exploited in biomedical practices. Began in the 1950 s, the differences between red and white blood cells' characteristic impedance variations due to their disparate sizes were employed for blood cell classification [11]. As impedance sensing techniques advanced, more correlations have been deeply explored in recent years. Here are several examples. The reduction in high-frequency impedance caused by antibiotics-induced bacterial morphology changes was found and then leveraged for the fast antimicrobial susceptibility test [12]. The impedance amplitude remarkable jumps induced by the abrupt reduction of cell volume were found and then exploited in real-time monitoring of single budding yeast cells' daughter dissection events [13], [14]. The association between the tilted impedance pulses and cell elongation/asymmetric shapes led by the paramylon's inhomogeneous distribution was used in the fatness characterization of Euglena gracilis cells [15]. In addition, the differentials between the same cells' native and deformed shape induced impedance magnitude variations were used for 'optics-free' characterization of single cells' deformability [16]. These studies all focused on the correlation finding under the same cell state─suspended state.

Similar correlations were beginning to be exploited in adherent cells. With current techniques, it is still difficult to quantify a single adherent cell's impedance phenotype in high-throughput. Nonetheless, the cell sheets' occupied area-induced impedance changes have been used to monitor adherence proliferation status [17] or evaluate drug response [18].

Previous studies preliminary explored the correlation between cells' morphologies and electrical properties for cell clusters or even single cells. But those works were all directly based on the same cell states, either adherent cell state or suspended state. Is it possible to extend this correlation to the two major different cell states? Is there any relationship between the adherent cell morphology and the electrical property after these adherent cells were resuspended? We think these questions are very interesting and worth being addressed. The single cells' adherent morphology before being suspended carries a wealth of biological information, such as tumor invasion abilities [4], [19], [20]. Cell shapes may strongly affect cellular function [21]. For example, Peng et al. reported that the subtype of both Huh7 and Hep3B cells with spindle shapes have higher invasion [22], and Yu et al. discovered that the specific spindle cell regions might be potential histomorphological characteristics of the sarcomatoid parathyroid carcinoma with high invasiveness [4]. But these pieces of biological information may be lost after the cells turn spherical-shaped in suspension. A link between the two important biophysical properties presented respectively in adherent and suspension states may help explore more comprehensive information for conventionally single-cell analysis in suspension. However, few studies have been reported in this field so far, and the questions remain unanswered.

In this paper, after systematically investigating the related literature, we found some clues for the questions mentioned above. Following those clues, for the first time, we experimentally found a quantitative correlation between the adherent cells' morphology and suspended cells' electrical properties at the single-cell level revealed from clinical samples in future exploration.

Table 1 listed the reported cell electrical properties (i.e., Csm, measured by various techniques), and the corresponding adherent cell shapes (estimated and concluded by this paper) for different cell lines. The clues were found in the summation of the reported data produced by different cellular electrical phenotyping methods. As listed in Table 1, five contrasting groups with their cells' electrical properties quantified by four different techniques were chosen. To avoid the misdirection caused by the between-group differences in the results quantified by different methods, we only focused on the variation tendency of the cell lines' cellular Csm and adherent morphology in the same group. Meanwhile, which was also worth being emphasized, as most reported electrical properties results have not been followed by their cellular adherent morphology, we only listed the cell lines whose adherent morphology was provided on the web pages (listed in the table) of American Type Culture Collection (ATCC). With the contrastive analysis of cells' two biophysical properties in every above group (Table 1), an apparent result between cellular electrical properties and adherent morphology emerged: all five round-shaped cell lines' cellular Csm were comparatively lower than their corresponding triangular- or spindle-shaped cell lines. This phenomenon suggests that the electrical properties of single cells which have been already suspended may "remember" their natural adherent morphology.

Although there have been clues listed above, it is still too early to conclude whether the correlation exists because the compared cells from tumor cell lines are all sampled from different persons suffering from different tumor types and might be high-passaged over tens of years [36]. In lung cancer patients' clinical pleural effusions (PEs), exfoliated cells have various morphologies that convey crucial tumor information [37], [38], [39], [40], [41]. Cells sampled from the same patient with a similar genetic background have the potential to be proliferated to comparative samples with different adherent morphologies to characterize their phenotypes further.

However, a substantial population contaminating blood cells and lymphocytes mingled with tiny amounts of rare large target cells exist in PEs [39], [42], giving rise to difficulties in enriching alive rare target cells from PEs without labeling. Furthermore, due to the interference from a myriad of background cells, primary PE cell culture in vitro was fiendishly intractable. Moreover, it is challenging to characterize thousands of collected rare cells' intrinsic electrical properties with high throughput, while it is also hard to quantify cells' adherent morphologies.

To overcome the above challenges, we proposed a toolchain for enrichment, proliferation, and quantification of two biophysical phenotypes of single primary cells from clinical PEs. For rare tumor cells enriching from PEs, the enrichment method is expected to be characterized by high throughput and high recovery. Long-term cell proliferation and culture in vitro required the enrichment method to be equipped to remove background cells while maintaining target cell viability. To meet the above requirements, we developed a 3D cell sieving method [43] and employed it here to enrich large-sized rare cells from PEs with high quality. The devised 3D sieving device effectively removed small cells (RBCs and lymphocytes) from PEs based on cell size, and the recovered cells were demonstrated with high proliferation capabilities. A crossing constriction channel-based impedance flow cytometry was developed [24] and used here to characterize cells' electrical properties with a high throughput of 100,000 cells/h.

Based on the above techniques, the entire workflow was proposed and illustrated in Fig. 1. Primitive PEs were collected from different lung adenocarcinoma patients (Fig. 1 A). The samples were then poured over the entrance of 3D sieving device to enrich target rare tumor cells (Fig. 1 B). Recovered cells were randomly seeded into several wells for culture in vitro, and then their adherent morphologies after proliferation were observed. Cells' round- or spindle-shaped adherent morphologies were quantified by the proposed elongation ratio (Er), the ratio between the minimum outer circle diameter Dout and the maximum inner circle diameter Din of the cell morphology outline (Fig. 1 C). With the developed impedance flow cytometry, the proliferated single cells were aspirated for continuously passing through the microfluidic crossing constriction channel with simultaneous impedance monitoring (Fig. 1 D). Combined with the equivalent electrical model for a single cell in the constriction channel (Fig. 1 E), the obtained impedance changes were translated into single-cell specific membrane capacitance Csm and cytoplasm conductivity σcyto (Fig. 1 F). Finally, the Er and Csm of 10 samples' adherent cells were characterized to explore their correlation in the following experiments.

Section snippets

Reagents

Unless otherwise stated, all reagents for cell culture were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Specifically, relevant reagents used in cell culture include RPMI-1640 Media (GIBCO, Life Technologies Corp., USA), Fetal Bovine Serum (GIBCO, Life Technologies Corp., USA), Phosphate Buffer Saline (PBS) (GIBCO, Life Technologies Corp., USA), Penicillin–Streptomycin (GIBCO, Life Technologies Corp., USA), 0.25% Trypsin (GIBCO, Life Technologies Corp., USA). 2-NBDG

Effective enrichment and proliferation of PEs cells

As the first step for cell sample preparation, the target cells were enriched from 50 mL of every patient's clinical PE and then proliferated to enough cells to be characterized. When exploring the performance of cell enrichment and proliferation, the PEs collected from patient #0 were initially used to evaluate cell enrichment and primary culture. The enrichment results (Fig. 2 A-C) show that the 3D cell sieve can potentially enrich tumor cells from large-volume clinical PE samples

Conclusion

Summarizing previously reported works, in their studied different types of tumor cell lines, we found that the Csm of single suspended cells with round-like shaped adherent morphology were generally lower than non-round. To verify whether this hypothesis holds in the cells with similar genetic backgrounds, we sampled primary cells from clinical patients' PEs, proliferated two different adherent morphologies, and characterized their single cells' electrical properties. The final statics analyses

CRediT authorship contribution statement

Xiaofeng Luan, Yang Zhao, Lina Zhang, Chengjun Huang, Haiping Zhao: Conceptualization, Experiments design, Writing the manuscript. Xiaofeng Luan, Yuang Li, Lina Zhang, Yang Zhao, Sheng Sun: Experiments, Data analysis. Xiaofeng Luan, Yang Zhao, Wenchang Zhang, Lingqian Zhang, Mingxiao Li, Yuanyuan Fan, Jinghui Wang, Tian Zhi: Visualization, Investigation, Data curation, Editing the manuscript. Lina Zhang, Yang Zhao, Chengjun Huang: Funding acquisition, Project administration.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by the National Key Research and Development Program of China (No. 2018YFC2001100), the National Natural Science Foundation of China (No. 62171441), the Scientific Research and Equipment Development Project of CAS (YJKYYQ20210031), State Key Laboratory of Computer Architecture (ICT, CAS) under Grant No. CARCH202122, and Youth Innovation Promotion Association of Chinese Academy of Sciences.

Xiaofeng Luan was born in Shandong, China, in May 1995. In 2018, she received her B.S. degree from the school of optical and electronic information, Huazhong University of Science and Technology, China. She is now a Ph.D. student of the Institute of Microelectronics, Chinese Academy of Sciences. Her research interests are single cell analysis, and microfluidic impedance flow cytometry.

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  • Cited by (0)

    Xiaofeng Luan was born in Shandong, China, in May 1995. In 2018, she received her B.S. degree from the school of optical and electronic information, Huazhong University of Science and Technology, China. She is now a Ph.D. student of the Institute of Microelectronics, Chinese Academy of Sciences. Her research interests are single cell analysis, and microfluidic impedance flow cytometry.

    Yuang Li was born in Shandong, China, in September 1997. In 2019, he received his B.S. degree from the school of electronics and information engineering, North China University of Technology, China. He is now a Ph.D. student of the Institute of Microelectronics, Chinese Academy of Sciences. His research interests are single cell analysis, and microfluidic cell sorting.

    Haiping Zhao received her Ph.D. degree from Peking Union Medical College in 2009. She is currently a researcher professor at the Laboratory of Cerebrovascular Diseases of Capital Medical University. Her current research mainly focuses on cerebral ischemia neuroprotection and translational medicine.

    Sheng Sun Sheng Sun was born in Beijing, China, in December 1997. In 2020, she received her B.S. degree from the school of electronic science and technology, Beijing University of Technology, China. She is now a Ph.D. student of the Institute of Microelectronics, Chinese Academy of Sciences. Her research interests are imaging flow cytometry, and single cell analysis.

    Yuanyuan Fan is an Associate Researcher in the Institute of Microelectronics of the Chinese Academy of Sciences (CAS). She received her Ph.D. at Shanghai Institute of Optics and Fine Mechanics, CAS. Her research is focused on the technology and application of laser, especially the infrared and ultraviolet light sources.

    Wenchang Zhang received his Ph.D. degree from Hefei University of Technology, China, in 2017. He is currently an associate professor at the Institute of Microelectronics of Chinese Academy of Sciences. His research is focused on microfluidic-based cell analysis and detection.

    Lingqian Zhang received her Ph.D. degree from the Institute of Microelectronics, Peking University in 2018. She is currently an associate professor at the Institute of Microelectronics of Chinese Academy of Sciences. Her current research mainly focuses on the micro-electromechanical systems (MEMS), Parylene-C based micro/nano fabrication, and bio-sensing.

    Mingxiao Li received his Ph.D. degree at the Department of Chemical Engineering, University of Louisville, USA. He obtained his bachelor degree in Chemical Engineering from Tsinghua University. He is currently a professor at the Institute of Microelectronics of Chinese Academy of Sciences. His research interests include nano/micro fabricated biosensors for rapid and sensitive detection of biomarkers for specific diseases.

    Jinghui Wang is an associate Professor of Medical Oncology, Beijing Chest Hospital, Capital Medical University. She received her M.D.in Beijing Tuberculosis and Thoracic Tumor Research Institute in 2014. Her research is focused on the personalized treatment for lung cancer.

    Tian Zhi is an Associate Professor of computer architecture at Institute of Computing, Chinese Academy of Sciences. She received his PhD at Institute of Electronics, Chinese Academy of Sciences in 2014. Her research is focused on the development of reconfigurable hardwares and Artificial intelligence algorithms.

    Lina Zhang is an assistant researcher of Cancer Research Center, Beijing Chest Hospital, Capital Medical University. She received her MD at Tianjin Medical University in 2007. Her research is focused on the circulating tumor cells in lung cancer metastasis and drug resistance.

    Yang Zhao received his Ph.D. degree from the Chinese Academy of Sciences in 2016. He is currently an associate professor at the Institute of Microelectronics of Chinese Academy of Sciences. His research interests include single-cell analysis and microfluidics.

    Chengjun Huang received his Ph.D. degree at Huazhong University of Science and Technology, China, in 2006. He is currently a professor at the Institute of Microelectronics of Chinese Academy of Sciences. His research interests include micro/nanosensors, microfluidics, and lab-on-a-chip.

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